Fluorometer and method for use in determining the composition of an analyte

Abstract

A fluorometer (2) and associated methods are disclosed for use in determining the composition of an analyte (4), for example for use in determining the composition of an analyte (4) comprising first, second and third constituents, and in particular though not exclusively, to a fluorometer and associated methods for use in assessing the quality of water comprising first, second and third constituents, wherein the first constituent comprises a first fluorescent constituent, the second constituent comprises a second fluorescent constituent, and the third constituent comprises a plurality of scattering particles. Such a fluorometer (2) and associated methods may be particularly suitable for water quality surveillance applications or in-situ water quality measurements at multiple different locations such as remote or inaccessible locations for extended periods of time. The fluorometer (2) includes first, second and third optical sources (6a, 6b, 6c) emitting first, second and third light (60a, 60b, 60c) for illuminating the analyte (4), wherein the first light (60a) has a first spectral range, the second light (60b) has a second spectral range, and the third light (60c) has a third spectral range. The fluorometer (2) also includes a photodetector (10) for detecting light output from the analyte (4) when the analyte (4) is illuminated by at least one of the first, second and third light (60a, 60b, 60c).

Description

[0001] FLUOROMETER AND METHOD FOR USE IN DETERMINING THE COMPOSITION OF AN ANALYTE

[0002] FIELD

[0003] The present disclosure relates to a fluorometer and associated methods for use in determining the composition of an analyte comprising first, second and third constituents, and in particular though not exclusively, to a fluorometer and associated methods for use in assessing the quality of water comprising first, second and third constituents, wherein the first constituent comprises a first fluorescent constituent, the second constituent comprises a second fluorescent constituent, and the third constituent comprises a plurality of scattering particles.

[0004] BACKGROUND

[0005] Tryptophan-like fluorescence (TLF) of water is known to be highly correlated with the presence of microbial contamination in the water. Humic-like fluorescence (HLF) of water is also known to be correlated with the presence of micro-organisms in the water and is known to indicate the dose required for disinfection such as chlorination. HLF is an important parameter in itself because humic matter, including humic acids, humin, fulvic acids due to contamination from the soil, is known to have health hazards, and produce disinfection by-products after reacting with disinfectants such as chlorine. Fluorescence properties of water including TLF, HLF and other excitation-emission pairs can provide an indication of the concentration of microplastics, fertilisers, algae, petroleum, phenols, microbial products, phenolic compounds, petroleum, surfactants, pharmaceutical compounds, or other chemicals in water. The turbidity of water - i.e. the concentration of suspended particles in water - is also an important parameter for drinking water. Consequently, to determine whether water is suitable for human consumption or suitable for other uses, it is known to measure TLF and HLF of water, and to measure the turbidity of the water.

[0006] Fluorometers are known which are capable of measuring the TLF and HLF of water for the determination of organic pollution levels in water. However, known fluorometers are not generally capable of measuring TLF and HLF in water in the presence of turbidity.

[0007] Moreover, fluorometers are known that require the use of highly sensitive photodetectors such as photomultiplier tubes (PMTs) for determining the concentration of a plurality of fluorescent species in water. Fluorometers are also known that require

[0008] 55789724-1the use of complex photodetector arrangements comprising multiple photodetectors and associated electronics, or a spectrum analyser, for determining the concentration of a plurality of fluorescent species in water.

[0009] Additionally or alternatively, known fluorometers may rely on the use of one or more spectral filters for spectrally filtering light emanating from a water sample before detecting the spectrally filtered light. For example, it is known to use one or more spectral filters for spectrally isolating fluorescence generated by a target fluorescent species in a water sample from fluorescence generated by other fluorescent species that may be present in the water sample and / or for spectrally isolating the fluorescence generated by the target fluorescent species in the water sample from any excitation light which is scattered by the water sample or which is reflected or transmitted by the water sample. Similarly, it is known to use one or more spectral filters for spectrally isolating excitation light which is scattered by the water sample or which is reflected or transmitted by the water sample from any fluorescence generated by one or more fluorescent species that may be present in the water sample. Such known fluorometers may have a fixed or dedicated spectral filter configuration which is selected according to the target fluorescent species or to the turbidity. For example, such known fluorometers may include one or more dedicated spectral detection channels, wherein each spectral detection channel comprises a corresponding optical detector and a corresponding fixed spectral filter. Additionally or alternatively, such known fluorometers may include a variable spectral detection channel having a variable spectral responsivity e.g. because the variable spectral detection channel comprises a variable spectral filter such as a movable spectral filter or a replaceable spectral filter.

[0010] Additionally or alternatively, known fluorometers may also comprise additional optical components such as beamsplitters to facilitate the measurement of different optical properties of a water sample such as fluorescence, transmission, absorption, and scattering.

[0011] For all of the foregoing reasons, known fluorometers may not be simple enough or robust enough for some applications. For example, known fluorometers may not be simple enough or robust enough for some water quality surveillance applications or in-situ water quality measurements which require multiple fluorometers to be deployed at multiple different locations for extended periods.

[0012] Additionally or alternatively, known fluorometers may be too large and / or too heavy for transportation to remote or inaccessible locations. Additionally or alternatively, known fluorometers may consume too much power to be used for water quality

[0013] 55789724-1surveillance applications or in-situ water quality measurements in remote or inaccessible locations for extended periods of time.

[0014] SUMMARY

[0015] According to an aspect of the present disclosure there is provided a fluorometer for use in determining the concentrations of first, second and third constituents of an analyte, wherein the first constituent comprises a first fluorescent constituent, the second constituent comprises a second fluorescent constituent, and the third constituent comprises a plurality of scattering particles, and wherein the fluorometer comprises: first, second and third optical sources for emitting first, second and third light with first, second and third spectral ranges respectively;

[0016] a photodetector for detecting light output from the analyte when the analyte is illuminated by at least one of the first, second and third light; and

[0017] a controller,

[0018] wherein the controller is configured to:

[0019] cause the first optical source to illuminate the analyte using the first light and cause the photodetector to detect light output from the analyte so as to generate a first electrical signal resulting from illumination of the analyte using the first light;

[0020] cause the second optical source to illuminate the analyte using the second light and cause the photodetector to detect light output from the analyte so as to generate a second electrical signal resulting from illumination of the analyte using the second light;

[0021] cause the third optical source to illuminate the analyte using the third light and cause the photodetector to detect light output from the analyte so as to generate a third electrical signal resulting from illumination of the analyte using the third light; and

[0022] determine a concentration of each of the first, second and third constituents based at least in part on the first, second and third electrical signals. Such a fluorometer may be used to determine the concentration of the first fluorescent constituent, the concentration of the second fluorescent constituent, and the concentration of scattering particles (i.e. the turbidity) to be determined in the analyte based at least in part on the first, second and third electrical signals measured using the same photodetector. The photodetector may for example be a single low-cost silicon PIN photodiode. Such a fluorometer may be simpler and more robust than known

[0023] 55789724-1fluorometers. Such a fluorometer may be smaller and lighter and may therefore be more portable than known fluorometers. Such a fluorometer may be more energy efficient than known fluorometers. Such a fluorometer may be more suitable for water quality surveillance applications or in-situ water quality measurements at multiple different locations such as remote or inaccessible locations for extended periods of time than known fluorometers.

[0024] Optionally, the light output from the analyte when the analyte is illuminated using the first light comprises fluorescence generated by the first fluorescent constituent and scattered first light, and optionally also fluorescence generated by the second fluorescent constituent.

[0025] Optionally, the light output from the analyte when the analyte is illuminated using the second light comprises fluorescence generated by the second fluorescent constituent and scattered second light, and optionally also fluorescence generated by the first fluorescent constituent.

[0026] Optionally, the light output from the analyte when the analyte is illuminated using the third light comprises scattered third light, and optionally also fluorescence generated by the first and / or second fluorescent constituents.

[0027] Optionally, the analyte is, or comprises, a liquid.

[0028] Optionally, the analyte comprises a base constituent such as water.

[0029] Optionally, the first, second and third constituents comprise first, second and third contaminants of the base constituent respectively.

[0030] Optionally, the first fluorescent constituent comprises a Tryptophan-like fluorescence (TLF)-emitting constituent.

[0031] Optionally, the second fluorescent constituent comprises a humic-like fluorescence (HLF)-emitting constituent.

[0032] Such a fluorometer may be used to measure the concentration of the TLF-emitting constituent, the concentration of the HLF-emitting constituent, and the concentration of the scattering particles (i.e. the turbidity) of the analyte. Such a fluorometer may be used to measure the concentration of the TLF-emitting constituent, the concentration of the HLF-emitting constituent, and the concentration of scattering particles (i.e. the turbidity) in the base constituent. In particular, such a fluorometer may be used to measure the concentration of a TLF-emitting constituent, the concentration of an HLF-emitting constituent, and the concentration of scattering particles (i.e. the turbidity) in water in order to determine whether the water is safe to drink or use, for example to determine whether the water complies with international safety standards

[0033] 55789724-1such as WHO standards for drinking water. Optionally, the first spectral range overlaps or falls within a first excitation spectral range of the first fluorescent constituent, the second spectral range overlaps or falls within a second excitation spectral range of the second fluorescent constituent, and the third spectral range falls outside the first and second excitation spectral ranges.

[0034] Optionally, the second spectral range partially overlaps or falls outside the first excitation spectral range of the first fluorescent constituent.

[0035] Optionally, the first spectral range of the first light is selected so that the first electrical signal generated by the photodetector when the analyte is illuminated using the first light is dependent on the concentration of the first, second and third constituents.

[0036] Optionally, the first spectral range of the first light is selected so that the first electrical signal generated by the photodetector when the analyte is illuminated using the first light is dependent on the concentration of the first and third constituents, but is independent of, or substantially independent of, the concentration of the second constituent.

[0037] Optionally, the second spectral range of the second light is selected so that the second electrical signal generated by the photodetector when the analyte is illuminated using the second light is dependent on the concentration of the second and third constituents, but independent of, or substantially independent of, the concentration of the first constituent.

[0038] Optionally, the third spectral range of the third light is selected so that the third electrical signal generated by the photodetector when the analyte is illuminated using the third light is dependent on the concentration of the third constituent, but independent of, or substantially independent of, the concentrations of the first and second constituents.

[0039] Optionally, the first excitation spectral range of the first fluorescent constituent has a first cut-off wavelength such that when the first fluorescent constituent is excited using light at wavelengths which are longer than the first cut-off wavelength, the first fluorescent constituent generates little or no fluorescence.

[0040] Optionally, the second excitation spectral range of the second fluorescent constituent has a second cut-off wavelength such that when the second fluorescent constituent is excited using light at wavelengths which are longer than the second cutoff wavelength, the second fluorescent constituent generates little or no fluorescence.

[0041] 55789724-1Optionally, the first spectral range includes a first peak wavelength, the second spectral range includes a second peak wavelength, and the third spectral range includes a third peak wavelength.

[0042] Optionally, the first peak wavelength falls within the first excitation spectral range, the second peak wavelength falls within the second excitation spectral range, and the third peak wavelength falls outside the first and second excitation spectral ranges.

[0043] Optionally, the second peak wavelength falls inside or outside the first excitation spectral range of the first fluorescent constituent.

[0044] Optionally, the first peak wavelength is shorter than the second peak wavelength, and the second peak wavelength is shorter than the third peak wavelength.

[0045] Optionally, the first and second excitation spectral ranges overlap in an overlap excitation spectral range at wavelengths less than the first cut-off wavelength and the first light has a first peak wavelength which is in the overlap excitation spectral range to cause both the first and second fluorescent constituents to generate fluorescence in the analyte and resulting in scattering of the first light in the analyte.

[0046] Optionally, the first and second excitation spectral ranges do not overlap and the first light has a first peak wavelength which is less than the first cut-off wavelength to cause the first fluorescent constituent to generate fluorescence in the analyte and resulting in scattering of the first light in the analyte, but without causing the second fluorescent constituent to generate fluorescence in the analyte.

[0047] Optionally, the second light has a second peak wavelength which is between the first and second cut-off wavelengths. Using such second light to illuminate the analyte may generate little or no fluorescence in the first fluorescent constituent but may generate fluorescence in the second fluorescent constituent and may result in scattering of the second light in the analyte.

[0048] Optionally, the third light has a third peak wavelength which is greater than the second cut-off wavelength. Using such third light to illuminate the analyte may generate little or no fluorescence in either the first or second fluorescent constituents, but may result in scattering of the third light in the analyte.

[0049] Optionally, the first spectral range is selected to excite TLF in the first fluorescent constituent.

[0050] Optionally, the first spectral range includes a peak TLF excitation wavelength. Optionally, the second spectral range is selected to excite HLF in the second fluorescent constituent.

[0051] Optionally, the second spectral range includes a peak HLF excitation wavelength.

[0052] 55789724-1Optionally, the third spectral range is selected to fall outside an excitation spectral range of TLF and outside an excitation spectral range of HLF.

[0053] Optionally, the first spectral range is selected to excite TLF in the first fluorescent constituent and HLF in the second fluorescent constituent.

[0054] Optionally, the second spectral range is selected to excite HLF in the second fluorescent constituent but not to excite TLF in the first fluorescent constituent.

[0055] Optionally, the first peak wavelength comprises a wavelength in the range of 265 to 295 nm, in the range of 275 to 285 nm, a wavelength of 280 nm or substantially equal to 280 nm, or a wavelength of 275 nm or substantially equal to 275 nm.

[0056] Optionally, the second peak wavelength comprises a wavelength in the range of 345 to 385 nm, in the range of 355 to 375 nm, or a wavelength of 365 nm or substantially equal to 365 nm.

[0057] Optionally, the third peak wavelength comprises a wavelength in the range of 500 to 1,100 nm, in the range of 900 to 1,000 nm, or a wavelength of 940 nm or substantially equal to 940 nm.

[0058] Optionally, the third peak wavelength comprises a wavelength in the range of 750 to 1,100 nm, in the range defined by the ISO:7027 specification, and / or in the range of 830 to 890 nm. Selecting a third peak wavelength in such wavelength ranges may at least partially the suppress the generation of fluorescence from the first and second fluorescent constituents that may occur from any fluorescent organic matter (e.g. protein, humic matter, chlorophyll, carotenoids) present in the analyte that may occur when illuminating the analyte using light in the UV-VIS region thereby improving the accuracy of the measurement of turbidity using the third light.

[0059] Optionally, one or more of the first, second and third optical sources comprises an LED or a laser diode.

[0060] Optionally, the photodetector comprises or is a photodiode.

[0061] Optionally, the photodetector comprises or is a single photodiode.

[0062] Optionally, the photodetector comprises or is a silicon photodiode.

[0063] Optionally, the photodetector comprises or is a PIN photodiode.

[0064] Optionally, the photodetector comprises or is a single pixel photodiode.

[0065] Optionally, the photodetector comprises an amplifier for amplifying an electrical signal generated by the photodiode.

[0066] Optionally, the amplifier comprises a multi-stage amplifier.

[0067] Optionally, the amplifier comprises a transimpedance stage and a gain stage. Optionally, the amplifier comprises a lock-in amplifier.

[0068] 55789724-1Optionally, the amplifier is configured to amplify electrical signals in a frequency range which includes the modulation frequency or frequencies of the first, second and third light.

[0069] Optionally, the amplifier is configured to only amplify electrical signals in a frequency range which includes the modulation frequency or frequencies of the first, second and third light.

[0070] Optionally, the amplifier is configured to amplify electrical signals in a frequency range of 250 Hz to 5 KHz.

[0071] Optionally, the amplifier is configured to only amplify electrical signals in a frequency range of 250 Hz to 5 KHz.

[0072] Optionally, the controller is configured to cause the first, second and third optical sources illuminate the analyte using the first, second and third light sequentially.

[0073] Optionally, the controller is configured to cause the first, second and third optical sources illuminate the analyte using the first, second and third light simultaneously.

[0074] Optionally, wherein at least one of:

[0075] the controller is configured to modulate the first light emitted by the first optical source and to cause the amplifier to use lock-in detection to generate the first electrical signal resulting from illumination of the analyte using the first light;

[0076] the controller is configured to modulate the second light emitted by the second optical source and to cause the amplifier to use lock-in detection to generate the second electrical signal resulting from illumination of the analyte using the second light; or the controller is configured to modulate the third light emitted by the third optical source and to cause the amplifier to use lock-in detection to generate the third electrical signal resulting from illumination of the analyte using the third light.

[0077] Optionally, the fluorometer comprises first, second and third drivers for electrically driving the first, second and third optical sources respectively.

[0078] Optionally, wherein at least one of:

[0079] the controller is configured to control the first driver to modulate the first light emitted by the first optical source and to cause the amplifier to use lock-in detection to generate the first electrical signal resulting from illumination of the analyte using the first light;

[0080] the controller is configured to control the second driver to modulate the second light emitted by the second optical source and to cause the amplifier to use lock-in detection to generate the second electrical signal resulting from illumination of the analyte using the second light; or

[0081] 55789724-1the controller is configured to control the third driver to modulate the third light emitted by the third optical source and to cause the amplifier to use lock-in detection to generate the third electrical signal resulting from illumination of the analyte using the third light.

[0082] Optionally, the controller is configured to modulate the first, second and third light differently.

[0083] Optionally, the controller is configured to amplitude modulate the first, second and third light at different frequencies.

[0084] Optionally, there are no spectral filters between the first optical source and the analyte.

[0085] Optionally, there are no spectral filters between the second optical source and the analyte.

[0086] Optionally, there are no spectral filters between the third optical source and the analyte.

[0087] Optionally, the fluorometer comprises at least one of:

[0088] one or more primary input optical elements between the first optical source and the analyte, wherein the one or more primary input optical elements are configured to couple the first light from the first optical source to the analyte;

[0089] one or more secondary input optical elements between the second optical source and the analyte, wherein the one or more secondary input optical elements are configured to couple the second light from the second optical source to the analyte; or one or more tertiary input optical elements between the third optical source and the analyte, wherein the one or more tertiary input optical elements are configured to couple the third light from the third optical source to the analyte.

[0090] Optionally, at least one of:

[0091] the one or more primary input optical elements comprise one or more primary lenses;

[0092] the one or more secondary input optical elements comprise one or more secondary lenses; or

[0093] the one or more tertiary input optical elements comprise one or more tertiary lenses.

[0094] Optionally, at least one of:

[0095] the one or more primary input optical elements comprise one or more primary windows;

[0096] 55789724-1the one or more secondary input optical elements comprise one or more secondary windows; or

[0097] the one or more tertiary input optical elements comprise one or more tertiary windows.

[0098] Optionally, the one or more primary windows are the only input optical elements between the first optical source and the analyte. This may allow the analyte to act as a heat sink for any heat generated by the first optical source thereby at least partially reducing the effects of heat on the spectra of the light output from the first optical source.

[0099] Optionally, the one or more secondary windows are the only input optical elements between the second optical source and the analyte. This may allow the analyte to act as a heat sink for any heat generated by the second optical source thereby at least partially reducing the effects of heat on the spectra of the light output from the second optical source.

[0100] Optionally, the one or more tertiary windows are the only input optical elements between the third optical source and the analyte. This may allow the analyte to act as a heat sink for any heat generated by the third optical source thereby at least partially reducing the effects of heat on the spectra of the light output from the third optical source.

[0101] Optionally, there are no spectral filters between the analyte and the photodetector.

[0102] Optionally, the fluorometer comprises one or more output optical elements between the analyte and the photodetector, wherein the one or more output optical elements are configured to couple light output from the analyte to the photodetector.

[0103] Optionally, the one or more output optical elements comprise one or more output lenses.

[0104] Optionally, the one or more output optical elements comprise one or more output windows.

[0105] Optionally, the one or more output windows are the only output optical elements between the analyte and the photodetector. This may allow the analyte to act as a heat sink and may at least partially reduce the effects of heat on the responsivity spectrum of the photodetector.

[0106] Optionally, at least two of the first, second and third optical sources are arranged to enable said at least two of the first, second and third optical sources to illuminate the analyte along the same input optical axis.

[0107] 55789724-1Optionally, at least two of the first, second and third optical sources are arranged side-by-side to enable said at least two of the first, second and third optical sources to illuminate the analyte along respective parallel input optical axes.

[0108] Optionally, at least two of the first, second and third optical sources are located at different positions to enable said at least two of the first, second and third optical sources to illuminate the analyte along respective non-parallel input optical axes.

[0109] Optionally, at least two of the first, second and third optical sources are arranged on opposite sides of the analyte.

[0110] Optionally, at least two of the first, second and third optical sources are arranged side-by-side on the same side of the analyte.

[0111] Optionally, the first optical source is arranged to illuminate the analyte along a first input direction, the second optical source is arranged to illuminate the analyte along a second input direction, the third optical source is arranged to illuminate the analyte along a third input direction, and the photodetector is arranged to receive light output from the analyte along an output direction, wherein the output direction is arranged at a non-zero angle relative to each of the first, second and third input directions.

[0112] Optionally, the output direction is arranged at an angle in the range of 85 to 95 degrees or in the range of 89 to 91 degrees, at an angle substantially equal to 90 degrees, or at an angle of 90 degrees relative to each of the first, second and third input directions.

[0113] Optionally, at least two of the first, second and third input directions are the same. Optionally, at least two of the first, second and third optical sources and the photodetector are located at different positions.

[0114] Optionally, the fluorometer comprises a user interface.

[0115] Optionally, the user interface comprises a display for displaying the determined concentration of each of the first, second and third constituents.

[0116] Optionally, the user interface comprises one or more user controls for controlling the operation of the fluorometer.

[0117] Optionally, the fluorometer comprises a battery for supplying electrical power to at least one of the first, second and third optical sources, the photodetector, the memory, and the controller.

[0118] Optionally, the battery is rechargeable.

[0119] Optionally, the fluorometer comprises a generator arrangement for recharging the battery.

[0120] Optionally, the generator arrangement comprises a solar cell or a solar panel.

[0121] 55789724-1Optionally, the fluorometer comprises a first Faraday cage, wherein the first Faraday cage encloses the photodetector.

[0122] Optionally, the fluorometer comprises a second Faraday cage, wherein the second Faraday cage encloses the first Faraday cage, and at least one of the first, second and third optical sources, the controller, and the memory.

[0123] Optionally, the fluorometer comprises a holder.

[0124] Optionally, the holder is configured for holding a container for containing the analyte.

[0125] Optionally, the fluorometer comprises the container for containing the analyte. Optionally, the container comprises a vessel, a vial or a cuvette.

[0126] Optionally, the container comprises a wall.

[0127] Optionally, the container wall is the only input optical element between the first optical source and the analyte. This may allow the analyte to act as a heat sink for any heat generated by the first optical source thereby at least partially reducing the effects of heat on the spectra of the light output from the first optical source.

[0128] Optionally, the container wall is the only input optical element between the second optical source and the analyte. This may allow the analyte to act as a heat sink for any heat generated by the second optical source thereby at least partially reducing the effects of heat on the spectra of the light output from the second optical source.

[0129] Optionally, the container wall is the only input optical element between the third optical source and the analyte. This may allow the analyte to act as a heat sink for any heat generated by the third optical source thereby at least partially reducing the effects of heat on the spectra of the light output from the third optical source.

[0130] Optionally, the container wall is the only output optical element between the analyte and the photodetector. This may allow the analyte to act as a heat sink and may at least partially reduce the effects of heat on the responsivity spectrum of the photodetector.

[0131] Optionally, the holder is configured for holding a flow cell or fluid conduit for conducting a flow of the analyte.

[0132] Optionally, the fluorometer comprises the flow cell or the fluid conduit for conducting the flow of the analyte.

[0133] Optionally, the flow cell or the fluid conduit comprises a wall.

[0134] Optionally, the wall of the flow cell or the fluid conduit is the only input optical element between the first optical source and the analyte. This may allow the analyte to act as a heat sink for any heat generated by the first optical source thereby at least

[0135] 55789724-1partially reducing the effects of heat on the spectra of the light output from the first optical source.

[0136] Optionally, the wall of the flow cell or the fluid conduit is the only input optical element between the second optical source and the analyte. This may allow the analyte to act as a heat sink for any heat generated by the second optical source thereby at least partially reducing the effects of heat on the spectra of the light output from the second optical source.

[0137] Optionally, the wall of the flow cell or the fluid conduit is the only input optical element between the third optical source and the analyte. This may allow the analyte to act as a heat sink for any heat generated by the third optical source thereby at least partially reducing the effects of heat on the spectra of the light output from the third optical source.

[0138] Optionally, the wall of the flow cell or the fluid conduit is the only output optical element between the analyte and the photodetector. This may allow the analyte to act as a heat sink and may at least partially reduce the effects of heat on the responsivity spectrum of the photodetector.

[0139] Optionally, the fluorometer comprises a housing, wherein the housing defines an interior space internally thereof for housing at least the first, second and third optical sources, the photodetector, the memory, and the controller.

[0140] Optionally, the housing defines an opening or an aperture for inserting the container into the interior space and / or for inserting the flow cell or fluid conduit into the interior space.

[0141] Optionally, the housing is configured so that the interior space is sealed from an environment exterior to the housing.

[0142] Optionally, the housing comprises a dipping section for dipping into the analyte, wherein the dipping section is configured to separate the first, second and third optical sources and the photodetector from the analyte when the dipping section is dipped into the analyte.

[0143] Optionally, the dipping section comprises at least one of:

[0144] one or more primary input optical elements for coupling the first light from the first optical source inside the housing to the analyte outside the housing;

[0145] one or more secondary input optical elements for coupling the second light from the second optical source inside the housing to the analyte outside the housing;

[0146] one or more tertiary input optical elements for coupling the third light from the third optical source inside the housing to the analyte outside the housing.

[0147] 55789724-1Optionally, at least one of:

[0148] the one or more primary input optical elements comprise one or more primary lenses;

[0149] the one or more secondary input optical elements comprise one or more secondary lenses; or

[0150] the one or more tertiary input optical elements comprise one or more tertiary lenses.

[0151] Optionally, at least one of:

[0152] the one or more primary input optical elements comprise one or more primary windows;

[0153] the one or more secondary input optical elements comprise one or more secondary windows; or

[0154] the one or more tertiary input optical elements comprise one or more tertiary windows.

[0155] Optionally, the one or more primary windows are the only input optical elements between the first optical source and the analyte.

[0156] Optionally, the one or more secondary windows are the only input optical elements between the second optical source and the analyte.

[0157] Optionally, the one or more tertiary windows are the only input optical elements between the third optical source and the analyte.

[0158] Optionally, the dipping section comprises one or more output optical elements for coupling light output from the analyte outside the housing to the photodetector inside the housing.

[0159] Optionally, the one or more output optical elements comprise one or more output lenses.

[0160] Optionally, the one or more output optical elements comprise one or more output windows.

[0161] Optionally, the one or more output windows are the only output optical elements between the analyte and the photodetector.

[0162] Optionally, the housing defines a space or a channel for the analyte externally thereof.

[0163] Optionally, the first, second and third optical sources and the photodetector are arranged around the space or the channel for the analyte.

[0164] Optionally, the first, second and third optical sources and the photodetector are arranged at one or more sides of the space or the channel for the analyte.

[0165] 55789724-1Optionally, each of the first, second and third optical sources and the photodetector are arranged at a different side of the space or the channel for the analyte.

[0166] Optionally, the space or the channel has a V-shape, a tetrahedral shape, or a pentahedral shape.

[0167] Optionally, the housing comprises a handle portion or is configured to be handheld.

[0168] Optionally, the fluorometer comprises a memory storing calibration data, wherein the controller is configured to determine the concentration of each of the first, second and third constituents based on the first, second and third electrical signals in combination with the stored calibration data. As will be described in more detail below, the calibration data may effectively define expected first, second and third electrical signals for any combination of known concentrations of the first, second and third constituents of a known analyte, thereby enabling unknown concentrations of the first, second and third constituents of an unknown analyte to be determined from measurements of the first, second and third electrical signals for the unknown analyte.

[0169] According to an aspect of the present disclosure there is provided a method for use in determining the concentrations of first, second and third constituents of an analyte, wherein the first constituent comprises a first fluorescent constituent, the second constituent comprises a second fluorescent constituent, and the third constituent comprises a plurality of scattering particles, and wherein the method comprises:

[0170] illuminating the analyte using first light having a first spectral range and using a photodetector to detect light output from the analyte so that the photodetector generates a first electrical signal resulting from illumination of the analyte using the first light; illuminating the analyte using second light having a second spectral range and using the photodetector to detect light output from the analyte so that the photodetector generates a second electrical signal resulting from illumination of the analyte using the second light;

[0171] illuminating the analyte using third light having a third spectral range and using the photodetector to detect light output from the analyte so that the photodetector generates a third electrical signal resulting from illumination of the analyte using the third light; and

[0172] determining a concentration of each of the first, second and third constituents based at least in part on the first, second and third electrical signals.

[0173] Such a method may enable the concentration of the first fluorescent constituent, the concentration of the second fluorescent constituent, and the concentration of

[0174] 55789724-1scattering particles (i.e. the turbidity) to be determined in the analyte based at least in part on the first, second and third electrical signals measured using the same photodetector. The photodetector may for example be a single low-cost silicon PIN photodiode. Such a method may be performed using a fluorometer which is simpler and more robust than known fluorometers. Such a method may be performed using a fluorometer which is smaller and lighter and which may therefore be more portable than known fluorometers. Such a method may be performed using a fluorometer which is more energy efficient than known fluorometers. Such a method may be performed using a fluorometer which is more suitable for water quality surveillance applications or in-situ water quality measurements at multiple different locations such as remote or inaccessible locations for extended periods of time than known fluorometers.

[0175] Optionally, the light output from the analyte when the analyte is illuminated using the first light comprises fluorescence generated by the first fluorescent constituent and scattered first light, and optionally also fluorescence generated by the second fluorescent constituent.

[0176] Optionally, the light output from the analyte when the analyte is illuminated using the second light comprises fluorescence generated by the second fluorescent constituent and scattered second light, and optionally also fluorescence generated by the first fluorescent constituent.

[0177] Optionally, the light output from the analyte when the analyte is illuminated using the third light comprises scattered third light, and optionally also fluorescence generated by the first and / or second fluorescent constituents.

[0178] Optionally, the analyte is, or comprises, a liquid.

[0179] Optionally, the analyte comprises a base constituent such as water, wherein the first, second and third constituents comprise first, second and third contaminants of the base constituent respectively.

[0180] Optionally, the first fluorescent constituent comprises a Tryptophan-like fluorescence (TLF)-emitting constituent.

[0181] Optionally, the second fluorescent constituent comprises a humic-like fluorescence (HLF)-emitting constituent.

[0182] Such a method may be used to measure the concentration of the TLF-emitting constituent, the concentration of the HLF-emitting constituent, and the concentration of the scattering particles (i.e. the turbidity) of the analyte. Such a method may be used to measure the concentration of the TLF-emitting constituent, the concentration of the HLF-emitting constituent, and the concentration of scattering particles (i.e. the turbidity) in the

[0183] 55789724-1base constituent. In particular, such a method may be used to measure the concentration of a TLF-emitting constituent, the concentration of an HLF-emitting constituent, and the concentration of scattering particles (i.e. the turbidity) in water in order to determine whether the water is safe to drink or use, for example to determine whether the water complies with international safety standards such as WHO standards for drinking water.

[0184] Optionally, the first spectral range overlaps a first excitation spectral range of the first fluorescent constituent, the second spectral range overlaps a second excitation spectral range of the second fluorescent constituent, and the third spectral range falls outside the first and second excitation spectral ranges.

[0185] Optionally, the first spectral range falls within the first excitation spectral range of the first fluorescent constituent, the second spectral range falls within the second excitation spectral range of the second fluorescent constituent, and the third spectral range falls outside the first and second excitation spectral ranges.

[0186] Optionally, the second spectral range partially overlaps or falls outside the first excitation spectral range of the first fluorescent constituent.

[0187] Optionally, the first spectral range of the first light is selected so that the first electrical signal generated by the photodetector when the analyte is illuminated using the first light is dependent on the concentration of the first, second and third constituents.

[0188] Optionally, the first spectral range of the first light is selected so that the first electrical signal generated by the photodetector when the analyte is illuminated using the first light is dependent on the concentration of the first and third constituents, but is independent of, or substantially independent of, the concentration of the second constituent.

[0189] Optionally, the second spectral range of the second light is selected so that the second electrical signal generated by the photodetector when the analyte is illuminated using the second light is dependent on the concentration of the second and third constituents, but independent of, or substantially independent of, the concentration of the first constituent.

[0190] Optionally, the third spectral range of the third light is selected so that the third electrical signal generated by the photodetector when the analyte is illuminated using the third light is dependent on the concentration of the third constituent, but independent of, or substantially independent of, the concentrations of the first and second constituents.

[0191] 55789724-1Optionally, the first excitation spectral range of the first fluorescent constituent has a first cut-off wavelength such that when the first fluorescent constituent is excited using light at wavelengths which are longer than the first cut-off wavelength, the first fluorescent constituent generates little or no fluorescence.

[0192] Optionally, the second excitation spectral range of the second fluorescent constituent has a second cut-off wavelength such that when the second fluorescent constituent is excited using light at wavelengths which are longer than the second cutoff wavelength, the second fluorescent constituent generates little or no fluorescence.

[0193] Optionally, the first spectral range includes a first peak wavelength, the second spectral range includes a second peak wavelength, and the third spectral range includes a third peak wavelength.

[0194] Optionally, the first peak wavelength falls within the first excitation spectral range, the second peak wavelength falls within the second excitation spectral range, and the third peak wavelength falls outside the first and second excitation spectral ranges.

[0195] Optionally, the second peak wavelength falls inside or outside the first excitation spectral range of the first fluorescent constituent.

[0196] Optionally, the first peak wavelength is shorter than the second peak wavelength, and the second peak wavelength is shorter than the third peak wavelength.

[0197] Optionally, the first and second excitation spectral ranges overlap in an overlap excitation spectral range at wavelengths less than the first cut-off wavelength and the first light has a first peak wavelength which is in the overlap excitation spectral range to cause both the first and second fluorescent constituents to generate fluorescence in the analyte and resulting in scattering of the first light in the analyte.

[0198] Optionally, the first and second excitation spectral ranges do not overlap and the first light has a first peak wavelength which is less than the first cut-off wavelength to cause the first fluorescent constituent to generate fluorescence in the analyte and resulting in scattering of the first light in the analyte, but without causing the second fluorescent constituent to generate fluorescence in the analyte.

[0199] Optionally, the second light has a second peak wavelength which is between the first and second cut-off wavelengths. Using such second light to illuminate the analyte may generate little or no fluorescence in the first fluorescent constituent but may generate fluorescence in the second fluorescent constituent and may result in scattering of the second light in the analyte.

[0200] Optionally, the third light has a third peak wavelength which is greater than the second cut-off wavelength. Using such third light to illuminate the analyte may generate little or

[0201] 55789724-1no fluorescence in either the first or second fluorescent constituents, but may result in scattering of the third light in the analyte.

[0202] Optionally, the first spectral range is selected to excite TLF in the first fluorescent constituent.

[0203] Optionally, the first spectral range includes a peak TLF excitation wavelength. Optionally, the second spectral range is selected to excite HLF in the second fluorescent constituent.

[0204] Optionally, the second spectral range includes a peak HLF excitation wavelength. Optionally, the third spectral range is selected to fall outside an excitation spectral range of TLF and outside an excitation spectral range of HLF.

[0205] Optionally, the first spectral range is selected to excite TLF in the first fluorescent constituent and HLF in the second fluorescent constituent.

[0206] Optionally, the second spectral range is selected to excite HLF in the second fluorescent constituent but not to excite TLF in the first fluorescent constituent.

[0207] Optionally, the first peak wavelength comprises a wavelength in the range of 265 to 295 nm, in the range of 275 to 285 nm, a wavelength of 280 nm or substantially equal to 280 nm, or a wavelength of 275 nm or substantially equal to 275 nm.

[0208] Optionally, the second peak wavelength comprises a wavelength in the range of 345 to 385 nm, in the range of 355 to 375 nm, or a wavelength of 365 nm or substantially equal to 365 nm.

[0209] Optionally, the third peak wavelength comprises a wavelength in the range of 500 to 1,100 nm, in the range of 900 to 1,000 nm, or a wavelength of 940 nm or substantially equal to 940 nm.

[0210] Optionally, the third peak wavelength comprises a wavelength in the range of 750 to 1,100 nm, in the range defined by the ISO:7027 specification, and / or in the range of 830 to 890 nm. Selecting a third peak wavelength in such wavelength ranges may at least partially the suppress the generation of fluorescence from the first and second fluorescent constituents that may occur from any fluorescent organic matter (e.g. protein, humic matter, chlorophyll, carotenoids) present in the analyte that may occur when illuminating the analyte using light in the UV-VIS region thereby improving the accuracy of the measurement of turbidity using the third light.

[0211] Optionally, the photodetector comprises a photodiode.

[0212] Optionally, the photodetector comprises a single photodiode.

[0213] Optionally, the photodetector comprises a silicon photodiode.

[0214] Optionally, the photodetector comprises a PIN photodiode.

[0215] 55789724-1Optionally, the photodetector comprises a single pixel photodiode.

[0216] Optionally, the steps of:

[0217] illuminating the analyte using first light and using the photodetector to detect light output from the analyte so as to generate a first electrical signal resulting from illumination of the analyte using the first light;

[0218] illuminating the analyte using second light and using the photodetector to detect light output from the analyte so as to generate a second electrical signal resulting from illumination of the analyte using the second light; and

[0219] illuminating the analyte using third light and using the photodetector to detect light output from the analyte so as to generate a third electrical signal resulting from illumination of the analyte using the third light,

[0220] are performed sequentially.

[0221] Optionally, the steps of:

[0222] illuminating the analyte using first light and using the photodetector to detect light output from the analyte so as to generate a first electrical signal resulting from illumination of the analyte using the first light;

[0223] illuminating the analyte using second light and using the photodetector to detect light output from the analyte so as to generate a second electrical signal resulting from illumination of the analyte using the second light; and

[0224] illuminating the analyte using third light and using the photodetector to detect light output from the analyte so as to generate a third electrical signal resulting from illumination of the analyte using the third light,

[0225] are performed simultaneously.

[0226] Optionally, wherein at least one of:

[0227] the first light is modulated and lock-in detection is used to generate the first electrical signal resulting from illumination of the analyte using the first light;

[0228] the second light is modulated and lock-in detection is used to generate the second electrical signal resulting from illumination of the analyte using the second light; or

[0229] the third light is modulated and lock-in detection is used to generate the third electrical signal resulting from illumination of the analyte using the third light.

[0230] Optionally, the first, second and third light are modulated differently.

[0231] Optionally, the first, second and third light are amplitude modulated at different frequencies.

[0232] 55789724-1Optionally, determining the concentration of each of the first, second and third constituents based at least in part on the first, second and third electrical signals comprises determining the concentration of each of the first, second and third constituents based on the first, second and third electrical signals in combination with calibration data.

[0233] As will be described in more detail below, the calibration data may effectively define expected first, second and third electrical signals for any combination of known concentrations of the first, second and third constituents of a known analyte, thereby enabling unknown concentrations of the first, second and third constituents of an unknown analyte to be determined from measurements of the first, second and third electrical signals for the unknown analyte.

[0234] Optionally, the calibration data are derived from:

[0235] measurements of the first electrical signal resulting from illumination using the first light of different known analytes having:

[0236] different known concentrations of the first constituent, and zero or insignificant concentrations of the second and third constituents;

[0237] different known concentrations of the second constituent, and zero or insignificant concentrations of the first and third constituents; and

[0238] different known concentrations of the third constituent, and zero or insignificant concentrations of the first and second constituents; measurements of the second electrical signal resulting from illumination using the second light of different known analytes having:

[0239] different known concentrations of the first constituent, and zero or insignificant concentrations of the second and third constituents;

[0240] different known concentrations of the second constituent, and zero or insignificant concentrations of the first and third constituents; and

[0241] different known concentrations of the third constituent, and zero or insignificant concentrations of the first and second constituents; and measurements of the third electrical signal resulting from illumination using the third light of different known analytes having:

[0242] different known concentrations of the first constituent, and zero or insignificant concentrations of the second and third constituents;

[0243] different known concentrations of the second constituent, and zero or insignificant concentrations of the first and third constituents; and

[0244] 55789724-1different known concentrations of the third constituent, and zero or insignificant concentrations of the first and second constituents. Optionally, the first spectral range is selected so that the first electrical signal resulting from illumination of the analyte using the first light is dependent on the concentration of the first, second and third constituents, the second spectral range is selected so that the second electrical signal resulting from illumination of the analyte using the second light is dependent on the concentration of the second and third constituents but independent of, or substantially independent of, the concentration of the first constituent, and the third spectral range is selected so that the third electrical signal resulting from illumination of the analyte using the third light is dependent on the concentration of the third constituent but independent of, or substantially independent of, the concentrations of the first and second constituents, and wherein the calibration data are derived from:

[0245] measurements of the first electrical signal resulting from illumination using the first light of different known analytes having:

[0246] different known concentrations of the first constituent, and zero or insignificant concentrations of the second and third constituents;

[0247] different known concentrations of the second constituent, and zero or insignificant concentrations of the first and third constituents; and

[0248] different known concentrations of the third constituent, and zero or insignificant concentrations of the first and second constituents; measurements of the second electrical signal resulting from illumination using the second light of different known analytes having:

[0249] different known concentrations of the second constituent, and zero or insignificant concentrations of the first and third constituents; and

[0250] different known concentrations of the third constituent, and zero or insignificant concentrations of the first and second constituents; and measurements of the third electrical signal resulting from illumination using the third light of different known analytes having:

[0251] different known concentrations of the third constituent, and zero or insignificant concentrations of the first and second constituents.

[0252] Optionally, the first spectral range is selected so that the first electrical signal resulting from illumination of the analyte using the first light is dependent on the concentration of the first and third constituents but independent of, or substantially independent of, the concentration of the second constituent, the second spectral range is selected so that the second electrical signal resulting from illumination of the analyte

[0253] 55789724-1using the second light is dependent on the concentration of the second and third constituents but independent of, or substantially independent of, the concentration of the first constituent, and the third spectral range is selected so that the third electrical signal resulting from illumination of the analyte using the third light is dependent on the concentration of the third constituent but independent of, or substantially independent of, the concentrations of the first and second constituents and

[0254] wherein the calibration data are derived from:

[0255] measurements of the first electrical signal resulting from illumination using the first light of different known analytes having:

[0256] different known concentrations of the first constituent, and zero or insignificant concentrations of the second and third constituents;

[0257] different known concentrations of the third constituent, and zero or insignificant concentrations of the first and second constituents; measurements of the second electrical signal resulting from illumination using the second light of different known analytes having:

[0258] different known concentrations of the second constituent, and zero or insignificant concentrations of the first and third constituents; and

[0259] different known concentrations of the third constituent, and zero or insignificant concentrations of the first and second constituents; and measurements of the third electrical signal resulting from illumination using the third light of different known analytes having:

[0260] different known concentrations of the third constituent, and zero or insignificant concentrations of the first and second constituents.

[0261] Optionally, the calibration data comprises responsivity coefficients m7and offset coefficients b^, wherein

[0262]

[0263] and h7are obtained by measuring the electrical signals s7= rrttj cj + bij for known concentrations cjof each constituent j excited by light i during a calibration procedure and using a regression analysis technique to fit the measured electrical signals s7as a function of the known concentrations cjof each constituent j, wherein i = 1,2,3 and j = 1,2,3.

[0264] Optionally, the regression analysis technique comprises a linear regression analysis technique.

[0265] Optionally, the regression analysis technique comprises a Multivariate Linear Regression analysis technique or a Bayesian regression analysis technique.

[0266] Optionally, determining the concentration of each of the first, second and third constituents based on the first, second and third electrical signals and the calibration

[0267] 55789724-1data comprises determining the concentration of each of the first, second and third constituents cjaccording to:

[0268] c = M-1(s — b)

[0269] wherein c is a concentration vector given by:

[0270] ci'

[0271] C2,

[0272]

[0273] C3.

[0274] s is an electrical signal vector given by:

[0275] -Si- S =S2,

[0276] S3.

[0277] st is the measured Ithelectrical signal generated by the photodetector resulting from illumination of the analyte using light i, M-1is the inverse of a responsivity matrix M given by:

[0278] [-mu "112 "113’

[0279] M = " I21 m22™23

[0280]

[0281] " I31 m32™33.

[0282] b is the offset vector given by:

[0283] V

[0284] t>2

[0285]

[0286] b̄₃

[0287] and b̄i=

[0288]

[0289] bij / 3 is the mean of the offset coefficients b7for the first, second and third constituents.

[0290] Optionally, the first spectral range, the second spectral range, and the third spectral range are selected relative to the first and second excitation spectral ranges of the first and second fluorescent constituents so that m2i= m31= m32= 0 i.e. so that:

[0291] mi2mi3- M =m22m23

[0292]

[0293] 0 ^33.

[0294] 55789724-1Optionally, the first spectral range, the second spectral range, and the third spectral range are selected relative to the first and second excitation spectral ranges of the first and second fluorescent constituents so that m21= m31= m32= m12= 0 i.e. so that:

[0295] mu 0 ^13'

[0296] 0 m22m23

[0297]

[0298] 0 0 m33.

[0299] It should be understood that any one or more of the features of any one of the foregoing aspects of the present disclosure may be combined with the features of any one or more of the other foregoing aspects of the present disclosure.

[0300] BRIEF DESCRIPTION OF THE DRAWINGS

[0301] A fluorometer and associated methods for determining the composition of an analyte comprising first, second and third constituents will now be described by way of non-limiting example only with reference to the drawings of which:

[0302] FIG. 1 is a block schematic of a fluorometer;

[0303] FIG. 2A is a schematic of the fluorometer of FIG. 1 illustrating the arrangement of features of the fluorometer within Faraday cages;

[0304] FIG. 2B is an exploded perspective view of the fluorometer of FIG. 1;

[0305] FIG. 3A is an exploded perspective view of an optical assembly of the fluorometer of FIG. 1;

[0306] FIG. 3B is a schematic plan view of the optical assembly of FIG. 3A;

[0307] FIG. 4A is a flowchart illustrating a calibration procedure for the fluorometer of FIG. 1;

[0308] FIG. 4B is a plot of the electrical signal measured by the fluorometer of FIG. 1 as a function of concentration of a known constituent during the calibration procedure shown in FIG. 4A;

[0309] 55789724-1FIG. 5A is a plot of the electrical signal measured by the fluorometer of FIG. 1 for different solutions of Tryptophan in water of different known concentrations during a calibration procedure when the different solutions are illuminated using first light;

[0310] FIG. 5B is a plot of the electrical signal measured by the fluorometer of FIG. 1 for different solutions of Quinine Sulphate in water of different known concentrations during a calibration procedure when the different solutions are illuminated using second light;

[0311] FIG. 50 is a plot of the electrical signal measured by the fluorometer of FIG. 1 for different solutions of Formazine in water of different known concentrations during a calibration procedure when the different solutions are illuminated using third light;

[0312] FIG. 6 is an exploded perspective view of an alternative optical assembly for use in the fluorometer of FIG. 1;

[0313] FIG. 7A is a side view of an alternative fluorometer;

[0314] FIG. 7B is an exploded perspective view of the fluorometer of FIG. 7A; and

[0315] FIG. 70 is a longitudinal cross-section of a dipping section of the fluorometer of FIG. 7A.

[0316] DETAILED DESCRIPTION OF THE DRAWINGS

[0317] Referring initially to FIG. 1 there is shown a schematic of a fluorometer generally designated 2 for determining the composition of a liquid analyte 4 comprising first, second, and third constituents, and a base constituent such as water, wherein the first constituent comprises a first fluorescent constituent, the second constituent comprises a second fluorescent constituent, and the third constituent comprises a plurality of scattering particles. The fluorometer 2 includes first, second and third optical sources for illuminating the analyte 4 in the form of first, second and third LEDs 6a, 6b and 6c for emitting first, second and third light 60a, 60b and 60c respectively, wherein the first light 60a has a first spectral range, the second light 60b has a second spectral range, and the third light 60c has a third spectral range. The fluorometer 2 further includes first, second and third LED drivers 8a, 8b, 8c for electrically driving the first, second and third optical sources 6a, 6b, 6c respectively. The fluorometer 2 also includes a photodetector

[0318] 55789724-1generally designated 10 including a silicon PIN photodiode 10a for detecting light output from the analyte 4 when the analyte 4 is illuminated by at least one of the first, second and third light 60a, 60b and 60c respectively. The photodetector 10 includes a multistage lock-in amplifier 10b for amplifying an electrical signal generated by the silicon PIN photodiode 10a, wherein the multi-stage lock-in amplifier 10b comprises a transimpedance stage 10b1 and a gain stage 10b2. In addition, the fluorometer 2 includes a user interface 12, a memory 14, and a controller in the form of a microcontroller 16. The memory 14 stores calibration data 18.

[0319] As shown in FIG. 2A, the fluorometer 2 further comprises a power supply 20 including a battery 22 and electronic circuitry (not shown explicitly in FIG. 2A) for controlling the supply of power from the battery 22 to the first, second and third LED drivers 8a, 8b, 8c, the photodetector 10, the user interface 12, the memory 14, and the controller 16. As shown in more detail in FIG. 3A, the photodetector 10 further comprises a circuit board 10c such as a PCB, wherein the amplifier 10b is mounted on the circuit board 10c. Referring back to FIG. 2A, the fluorometer 2 further comprises a first housing 24 enclosing the photodiode 10a, the multi-stage amplifier 10b and the circuit board 10c. The first housing 24 is configured to act as a first Faraday cage for at least partially suppressing any noise induced in the electrical signal generated by the photodetector 10, for example due to any electromagnetic interference resulting from operation of the first, second and third LED drivers 8a, 8b, 8c, the user interface 12, the memory 14, the controller 16, or the power supply 20. The fluorometer 2 further comprises a second housing 26 enclosing the first, second and third LEDs 6a, 6b and 6c, the first, second and third LED drivers 8a, 8b, 8c, the photodetector 10, the user interface 12, the memory 14, the controller 16, the power supply 20, and the first housing 24. In this regard, it should be understood that the second and third LEDs 6b and 6c, and the second and third LED drivers 8b, 8c are not shown in FIG. 2A in the interests of clarity. The second housing 26 is configured to act as a second Faraday cage for at least partially suppressing any noise induced in the electrical signal which is generated by the photodetector 10 and transferred to the controller 16, for example due to any electromagnetic interference originating from an environment external to the second housing 26.

[0320] As shown in FIG. 2B, the user interface 12 comprises a display 12a, one or more indicator LEDs 12b, one or more user controls 12c for controlling the operation of the fluorometer 2, and associated electronic circuitry (not shown explicitly in FIG. 2B). The fluorometer 2 further comprises an optical assembly generally designated 30. As will be

[0321] 55789724-1described in more detail below, the optical assembly 30 includes the first, second, and third LEDs 6a, 6b, 6c, the photodetector 10, and other optical elements (not shown in FIG. 2B) for coupling light from the first, second and third LEDs 6a, 6b and 6c to a container 31 such as a cuvette for containing the analyte 4, and for coupling light from the container 31 to the photodetector 10. The fluorometer 2 further includes a circuit board 40 such as a PCB on which the first, second and third LED drivers 8a, 8b, 8c, the memory 14, the controller 16, and the electronic circuitry of the power supply 20 are mounted. As shown in FIG. 2B, the second housing 26 includes first, second, and third parts 26a, 26b, and 26c which may be assembled together to house the battery 22, the circuit board 40, the optical assembly 30 and the electronic circuitry of the user interface 12.

[0322] As shown in more detail in FIG. 3A, the optical assembly 30 includes a holder in the form of an analyte block 32, a first input block 34, a second input block 36, and an output block 38. The first input block 34, the second input block 36, and the output block 38 are configured for attachment to different sides of the analyte block 32 using one or more fasteners in the form of one or more bolts or screws 39. The first and second LEDs 6a, 6b are attached side-by-side to the first input block 34. The third LED 6c is attached to the second input block 36 on the opposite side of the container 31 to the first and second LEDs 6a, 6b. The first housing 24 enclosing the photodetector 10 is attached to the output block 38.

[0323] As shown in FIG. 3B, the analyte block 32 is configured to hold the container 31 for the analyte 4. The first input block 34 holds one or more primary input optical elements in the form of one or more primary input lenses 44 for coupling light from first and second LEDs 6a, 6b to the analyte 4 in the container 31. The second input block 36 holds one or more secondary input optical elements in the form of one or more secondary input lenses 46 for coupling light from third LED 6c to the analyte 4 in the container 31. The output block 38 holds one or more output optical elements in the form of one or more output lenses 48 for coupling light from the analyte 4 in the container 31 to the photodiode 10a.

[0324] As shown in FIG. 3B, the optical assembly 30 is configured so that the first and second LEDs 6a and 6b are located side-by-side on a first input side of the container 31 for illuminating the analyte 4 in the container 31 along a first input direction and the third LED 6c is located on a second input side of the container 31 for illuminating the analyte 4 in the container 31 along a second input direction, wherein the second input side of the container 31 is opposite to the first input side of the container 31 and the second input

[0325] 55789724-1direction is opposite to the first input direction. The photodiode 10a is located on an output side of the container 31 so as to receive light output from the analyte 4 in the container 31 along an output direction, wherein the output direction is perpendicular to the first and second input directions so as to minimise the amount of scattered light detected by the photodiode 10a, wherein the scattered light comprises light from the first, second and third LEDs 6a, 6b, 6c which is scattered in the analyte 4.

[0326] In use, the controller 16 controls the first, second and third LED drivers 8a, 8b, 8c sequentially so that the first, second and third LEDs 6a, 6b, 6c emit modulated first, second and third light 60a, 60b, 60c during different time periods and the amplifier 10b uses lock-in detection to amplify the electrical signals generated by the photodiode 10a during the different time periods to generate first, second and third electrical signals respectively. Specifically, during a first time period, the controller 16 controls the first LED driver 8a to modulate the first light 60a emitted by the first LED 6a, the controller 16 de-activates the second and third LEDs 6b, 6c, and the controller 16 causes the amplifier 10b to use lock-in detection to amplify the electrical signal generated by the photodiode 10a to thereby generate the first electrical signal resulting from illumination of the analyte 4 using the first light 60a. Similarly, during a second time period, the controller 16 controls the second LED driver 8b to modulate the second light 60b emitted by the second LED 6b, the controller 16 de-activates the first and third LEDs 6a, 6c, and the controller 16 causes the amplifier 10b to use lock-in detection to amplify the electrical signal generated by the photodiode 10a to thereby generate the second electrical signal resulting from illumination of the analyte 4 using the second light 60b. In addition, during a third time period, the controller 16 controls the third LED driver 8c to modulate the third light 60c emitted by the third LED 6c, the controller 16 de-activates the first and second LEDs 6a, 6b, and the controller 16 causes the amplifier 10b to use lock-in detection to amplify the electrical signal generated by the photodiode 10a to thereby generate the third electrical signal resulting from illumination of the analyte 4 using the third light 60c. One of skill in the art will understand that the amplifier 10b is configured to only amplify electrical signals in a frequency range which includes the modulation frequency of the first, second and third light. For example, the first, second and third light 60a, 60b, 60c may be modulated at a frequency in a modulation frequency range of 250 Hz to 5 KHz and the amplifier 10b may comprise a low-noise amplifier which is configured to only amplify electrical signals in the modulation frequency range of 250 Hz to 5 KHz. One of skill in the art will understand that the use of amplitude modulation and lock-in detection

[0327] 55789724-1may reduce background noise detected by the photodetector 10 and improve the signal-to-noise ratio of the first, second and third electrical signals.

[0328] As will be described in more detail below, the controller 16 then determines a concentration of each of the first, second and third constituents of the analyte 4 based on the first, second and third electrical signals and the calibration data 18 stored in the memory 14.

[0329] FIGS. 4A and 4B illustrate a multispectral calibration procedure performed using a series of analytes of known composition to determine the calibration data 18. As shown in FIG. 4A, the controller 16 controls the first, second and third LED drivers 8a, 8b, 8c to modulate the light emitted by the IthLED of the first, second and third LEDs 6a, 6b, 6c and to de-activate the other two of the first, second and third LEDs 6a, 6b, 6c. The controller 16 reads a value stj of the electrical signal generated by the photodetector 10 resulting from illumination of the analyte 4 using the light emitted from the activated IthLED for different known concentrations cjof the1constituent in the analyte 4 and zero or insignificant concentrations of the other two of the first, second and third constituents. The controller 16 uses linear regression to fit the measured electrical signal values s7as a function of the known concentrations cjof the f constituent according to the equation s7= m7cj+ bijand determines the slope or responsivity coefficient m7and the intercept or offset coefficient h7as illustrated in FIG. 4B. The controller 16 then repeats the measurement of the electrical signal values s7as a function of the known concentrations cjof the1constituent for all relevant combinations of i and j, where i = 1,2,3 and j = 1,2,3.

[0330] The controller 16 models the electrical signal generated by the photodetector 10 as a result of illumination of the analyte 4 using the light emitted from the activated IthLED is modelled as:

[0331] st rriijCj + bL

[0332]

[0333] where, bL=

[0334]

[0335] bij / 3 is the mean of the offset coefficients bLj. The above equation can be expressed in a matrix form:

[0336] s = Me + b

[0337] wherein s is an electrical signal vector given by:

[0338] 55789724-1S =

[0339]

[0340] c is the concentration vector given by:

[0341] CT

[0342] c = c2

[0343] c3.

[0344] the responsivity matrix M is given by:

[0345] "112 "113’

[0346] M = m₂₁ m₂₂ m₂₃

[0347]

[0348] m₃₁ m₃₂ m₃₃

[0349] and the offset vector b is given by:

[0350] V

[0351] b = b̄₂

[0352]

[0353] b̄₃

[0354] The offset terms

[0355]

[0356] represent the fluorescence / scatter signal at zero concentration of compound j due to excitation by the LED i. Ideally, bijis independent of the compound j and depends only on the type of excitation i.e. bijdepends only on i.

[0357] From the foregoing description, one of skill in the art will understand that the responsivity matrix M and the offset vector b are determined during the calibration procedure and together constitute the calibration data 18 which is used to predict the electrical signal value generated when any one of the first, second and third LEDs 6a, 6b, 6c is used to illuminate the analyte 4 having any combination of known concentrations of the first, second and third constituents. Conversely, the unknown concentrations cjof each of the first, second and third constituents of the analyte 4 may be determined from the measured first, second and third electrical signals in combination with the responsivity matrix M and the offset vector b of the calibration data 18 according to:

[0358] c = M-1(s − b)

[0359] 55789724-1wherein M-1is the inverse of the responsivity matrix M.

[0360] From the foregoing description one of skill in the art will understand that the concentration of the first fluorescent constituent, the concentration of the second fluorescent constituent c2, and the concentration of scattering particles (i.e. the turbidity) c3of the analyte 4 may be determined based on measurements of the first, second and third electrical signals s1, s2, and s3and the calibration data 18 stored in the memory 14.

[0361] One of skill in the art will understand that the above method for determining the concentrations of the first fluorescent constituent, the second fluorescent constituent, and the plurality of scattering particles, relies upon the use of a single simple photodetector such as a single low-cost silicon PIN photodiode. Moreover, the above method for determining the concentrations of the first fluorescent constituent, the second fluorescent constituent, and the plurality of scattering particles comprises detecting the fluorescence and / or scattered light emanating simultaneously from the first fluorescent constituent, the second fluorescent constituent, and the plurality of scattering particles in the analyte at different wavelengths using the same photodetector when the analyte is illuminated using light from each of the LEDs 6a, 6b, 6c. Such a method does not rely on the use of any spectral filters between any of the LEDs 6a, 6b, 6c and the analyte 4 or between the analyte 4 and the photodiode 10a to spectrally discriminate between fluorescence emanating from different fluorescent constituents and the scattered excitation light. In particular, the above method for determining the concentrations of the first fluorescent constituent, the second fluorescent constituent, and the plurality of scattering particles does not rely on the use of any fixed or dedicated spectral filters or the use of any variable (e.g. movable or replaceable) spectral filters. Furthermore, the above method for determining the concentrations of the first fluorescent constituent, the second fluorescent constituent, and the plurality of scattering particles does not rely upon the use of any beamsplitter components between any of the LEDs 6a, 6b, 6c and the analyte 4 or between the analyte 4 and the photodiode 10a. For all of these reasons, such a method may be performed using a fluorometer which is simpler and more robust than known fluorometers and which is therefore more suitable for water quality surveillance applications or in-situ water quality measurements at multiple different locations such as remote or inaccessible locations for extended periods of time than known fluorometers. Such a method may be performed using a fluorometer which is smaller and lighter and which is therefore more portable than known fluorometers and which is therefore more suitable for water quality surveillance applications or in-situ water quality measurements at multiple different locations such as remote or inaccessible

[0362] 55789724-1locations for extended periods of time than known fluorometers. Such a method may be performed using a fluorometer which is more energy efficient than known fluorometers and which is therefore more suitable for water quality surveillance applications or in-situ water quality measurements at multiple different locations such as remote or inaccessible locations for extended periods of time than known fluorometers.

[0363] One of skill in the art will understand that the calibration procedure described with reference to FIGS. 4A and 4B represents a general case, wherein the electrical signal generated when illuminating the analyte 4 with any one of the first, second and third LEDs 6s, 6b, 6c is dependent on the concentration of any one of the first, second and third constituents. In practice, however, the electrical signal generated when illuminating the analyte 4 with one of the first, second and third LEDs 6s, 6b, 6c may be independent, or substantially independent, of the concentration of one or more of the first, second and third constituents i.e. one or more of the responsivity coefficients mijof the responsivity matrix M may be zero, or negligible relative to the other responsivity coefficients mij

[0364]

[0365] ijof the responsivity matrix M.

[0366] For example, the first spectral range of the first LED 6a may be selected to overlap with a first excitation spectral range of the first fluorescent constituent, the second spectral range of the second LED 6b may be selected to overlap with a second excitation spectral range of the second fluorescent constituent, and the third spectral range of the third LED 6c may be selected so as to fall outside the first and second excitation spectral ranges. The first spectral range of the first LED 6a may include a first peak wavelength, the second spectral range of the second LED 6b may include a second peak wavelength, and the third spectral range of the third LED 6c may include a third peak wavelength. The first peak wavelength may be selected to be equal to, or near, a peak fluorescence excitation wavelength of the first fluorescent constituent. The second peak wavelength may be selected to be equal to, or near, a peak fluorescence excitation wavelength of the second fluorescent constituent. The third peak wavelength may be selected to be sufficiently removed from the first and second excitation spectral ranges that the third spectral range of the third LED 6c may be considered to fall outside the first and second excitation spectral ranges. The first peak wavelength may be shorter than the second peak wavelength, and the second peak wavelength may be shorter than the third peak wavelength.

[0367] Moreover, the first excitation spectral range of the first fluorescent constituent may have a first cut-off wavelength such that when the first fluorescent constituent is excited using light at wavelengths which are longer than the first cut-off wavelength, the

[0368] 55789724-1first fluorescent constituent generates little or no fluorescence. Similarly, the second excitation spectral range of the second fluorescent constituent may have a second cutoff wavelength such that when the second fluorescent constituent is excited using light at wavelengths which are longer than the second cut-off wavelength, the second fluorescent constituent generates little or no fluorescence. Furthermore, the first excitation spectral range and the second excitation spectral range may overlap in an overlap excitation spectral range at wavelengths less than the first cut-off wavelength. Under these circumstances, the inventors have realised that illumination of the analyte 4 with first light 60a having a first peak wavelength which is in the overlap excitation spectral range will cause both the first and second fluorescent constituents to generate fluorescence in the analyte 4 and will be subject to scattering in the analyte 4; illumination of the analyte 4 with second light 60b having a second peak wavelength which is between the first and second cut-off wavelengths will generate little or no fluorescence in the first fluorescent constituent but will generate fluorescence in the second fluorescent constituent, and will be subject to scattering in the analyte 4; and illumination of the analyte 4 with third light 60c having a third peak wavelength which is greater than the second cut-off wavelength will generate little or no fluorescence in either the first or second fluorescent constituents, but will be subject to scattering in the analyte 4. Put another way, the first spectral range of the first light 60a may be selected so that the first electrical signal s1generated by the photodetector 10 when the analyte 4 is illuminatedusing the first light 60a is dependent on the concentration of the first, second and third constituents, the second spectral range of the second light 60b may be selected so that the second electrical signal s2generated by the photodetector 10 when the analyte 4 is illuminated using the second light 60b is dependent on the concentration of the second and third constituents, but independent of, or substantially independent of, the concentration of the first constituent (i.e. m21= 0), and the third spectral range of the third light 60c is selected so that the third electrical signal s3generated by the photodetector 10 when the analyte 4 is illuminated using the third light 60c is dependent on the concentration of the third constituent, but independent of, or substantially independent of, the concentrations of the first and second constituents (i.e. m31= m32= 0), resulting in a simplified responsivity matrix M of the form:

[0369] m11m12m13M = 0 m22m23

[0370]

[0371] 0 0 m33

[0372] 55789724-1Under these circumstances, the calibration procedure may be simplified to determine only the non-zero responsivity coefficients

[0373]

[0374] of the simplified responsivity matrix M.

[0375] For example, for a first fluorescent constituent which emits Tryptophan-like fluorescence (TLF) having a peak fluorescence excitation wavelength around 280 nm and a peak fluorescence emission wavelength around 350 nm, the first peak wavelength may be selected to be 275 nm. For a second fluorescent constituent which emits humic-like fluorescence (HLF) having a peak fluorescence excitation wavelength around 360 nm and a peak fluorescence emission wavelength around 450 nm, the second peak wavelength may be selected to be 365 nm. The third peak wavelength may be selected to be sufficiently removed from the first and second excitation spectral ranges associated with TLF and HLF that the third spectral range of the third LED 6c may be considered to fall outside the first and second excitation spectral ranges associated with TLF and HLF. For example, the third peak wavelength may be selected to be 940 nm.

[0376] Moreover, it should be understood that the first excitation spectral range and the second excitation spectral range may partially overlap. For example, TLF may be excited using an excitation wavelength in a first excitation spectral range below around 300 nm, whereas HLF may be excited over a wider second excitation spectral range which overlaps with the first excitation spectral range of TLF below around 300 nm but which extends above 300 nm up to a wavelength above 365 nm. Consequently, the inventors have realised that in the event that the analyte 4 comprises first, second and third constituents, wherein the first constituent comprises a species which is capable of emitting TLF, the second constituent comprises a species which is capable of emitting HLF, and the third constituent comprises a plurality of scattering particles, illumination of the analyte 4 with first light 60a having a first peak wavelength of 275 nm excites TLF and HLF in the analyte 4 and will be subject to scattering in the analyte 4; illumination of the analyte 4 with second light 60b having a second peak wavelength of 365 nm will not excite TLF in the analyte 4, but will excite HLF in the analyte 4, and will be subject to scattering in the analyte 4; and illumination of the analyte 4 with third light 60c having a third peak wavelength of 940 nm will excite neither TLF nor HLF in the analyte 4, but will be subject to scattering in the analyte 4. Put another way, the second electrical signal s2generated by the photodetector 10 when the analyte 4 is illuminated by the second light 60b having a second peak wavelength of 365 nm is independent of the concentration of the first fluorescent constituent (i.e. m21= 0) and the third electrical signal s3generated by the photodetector 10 when the analyte 4 is illuminated by the third

[0377] 55789724-1light 60c having a third peak wavelength of 940 nm is independent of the concentrations of the first and second fluorescent constituents (i.e. m31= m32= 0), resulting in a simplified responsivity matrix M of the form:

[0378] m11m12m13m22m23

[0379]

[0380] 0 0 m33

[0381] Consequently, when the first constituent comprises a species which is capable of emitting TLF, the second constituent comprises a species which is capable of emitting HLF, the third constituent comprises a plurality of scattering particles, and the first, second and third peak wavelengths are selected to be 275 nm, 365 nm, and 940 nm, the calibration procedure may be simplified to determine only the non-zero responsivity coefficients m^ of the simplified responsivity matrix M as will be described below with reference to FIGS. 5A to 5C.

[0382] FIG. 5A shows values of the measured electrical signal s1generated by the photodetector 10 when the analyte 4 is illuminated by the first light 60a having a first peak wavelength of 275 nm as a function of known concentrations of a first fluorescent constituent which is capable of emitting TLF (in the form of solutions of Tryptophan in water of different known concentrations), and for a zero or insignificant concentration of a second fluorescent constituent which is capable of emitting HLF, and for a zero or insignificant concentration of scattering particles. Linear regression is used to fit the data shown in FIG. 5A and a value of mu is determined from the slope of the resulting linear regression line. Similarly, the electrical signal s1generated by the photodetector 10 is also measured when the analyte 4 is illuminated by the first light 60a having a first peak wavelength of 275 nm as a function of known concentrations of a second fluorescent constituent which is capable of emitting HLF (in the form of solutions of Quinine Sulphate in water of different known concentrations), and for a zero or insignificant concentration of the first fluorescent constituent which is capable of emitting TLF, and for a zero or insignificant concentration of scattering particles. Linear regression is used to fit the resulting measurement data and a value of m12is determined from the slope of the resulting linear regression line. The electrical signal s1generated by the photodetector 10 is also measured when the analyte 4 is illuminated by the first light 60a having a first peak wavelength of 275 nm as a function of known concentrations of scattering particles (in the form of solutions of different known concentrations of Formazine in water), and for a zero or insignificant concentration of the first fluorescent constituent which is

[0383] 55789724-1capable of emitting TLF, and for a zero or insignificant concentration of the second fluorescent constituent which is capable of emitting HLF. Linear regression is used to fit the resulting measurement data and a value of m13is determined from the slope of the resulting linear regression line.

[0384] FIG. 5B shows values of the measured electrical signal s2generated by the photodetector 10 when the analyte 4 is illuminated by the second light 60b having a second peak wavelength of 365 nm as a function of known concentrations of a second fluorescent constituent which is capable of emitting HLF (in the form of solutions of Quinine Sulphate in water of different known concentrations), and for a zero or insignificant concentration of a first fluorescent constituent which is capable of emitting TLF, and for a zero or insignificant concentration of scattering particles. Linear regression is used to fit the data shown in FIG. 5B and a value of m22is determined from the slope of the resulting linear regression line. The electrical signal s2generated by the photodetector 10 is also measured when the analyte 4 is illuminated by the second light 60b having a first peak wavelength of 365 nm as a function of known concentrations of scattering particles (in the form of solutions of different known concentrations of Formazine in water), and for a zero or insignificant concentration of the first fluorescent constituent which is capable of emitting TLF, and for a zero or insignificant concentration of the second fluorescent constituent which is capable of emitting HLF. Linear regression is used to fit the resulting measurement data and a value of m23is determined from the slope of the resulting linear regression line.

[0385] FIG. 5C shows values of the measured electrical signal s3generated by the photodetector 10 when the analyte 4 is illuminated by the third light 60c having a third peak wavelength of 940 nm as a function of known concentrations of scattering particles (in the form of solutions of different known concentrations of Formazine in water) for a zero or insignificant concentration of a first fluorescent constituent which is capable of emitting TLF and for a zero or insignificant concentration of a second fluorescent constituent which is capable of emitting HLF. Linear regression is used to fit the data shown in FIG. 5C and a value of m33is determined from the slope of the resulting linear regression line.

[0386] From the foregoing description, one of skill in the art will understand that by selecting the first, second and third peak wavelengths to be 275 nm, 365 nm, and 940 nm, the fluorometer 2 may be calibrated and then used to determine the concentration of a first TLF-emitting constituent in the analyte 4, the concentration of a second HLF-emitting constituent in the analyte 4, and the concentration of scattering particles in the

[0387] 55789724-1analyte 4 i.e. the turbidity of the analyte 4. In particular, such a method may be used to measure the TLF, HLF and the turbidity of water in order to determine whether the water is safe to drink or use, for example to determine whether the water complies with international safety standards such as WHO standards for drinking water.

[0388] Moreover, the fluorometer 2 and the associated method of determining the concentration of a first TLF-emitting constituent, the concentration of a second HLF-emitting constituent and the concentration of scattering particles in the analyte 4 rely on the use of a single simple photodiode such as a single silicon PIN photodiode. Consequently, the fluorometer 2 may be simple enough and robust enough for water quality surveillance applications or in-situ water quality measurements which require multiple fluorometers to be deployed at multiple different locations such as remote or inaccessible locations for extended periods of time. The fluorometer 2 may also be smaller, lighter, and / or more power efficient than known fluorometers.

[0389] Rather than the analyte 4 being contained in a container 31 such as a cuvette, the analyte 4 may flow past the first, second and third light 60a, 60b, 60c within a flow cell or fluid conduit. For example, as shown in FIG. 6, the analyte block 32 may be configured for holding a flow cell 33 and multiple different sections of tubing 35a, 35b, and 35c may be used to connect the flow cell 33 to other fluid flow components such as a valve 37 and a fluid pump (not shown). When closed, the valve 37 may maintain static fluid conditions in the flow cell 33 for a consistent set of measurements of the analyte 4 in the flow cell 33.

[0390] Referring now to FIGS. 7A to 7C, there is shown an alternative fluorometer in the form of a hand-held fluorometer generally designated 102 for dipping into an analyte 4 and determining the composition of the analyte 4. Like the fluorometer 2 described with reference to FIGS. 2A, 2B, 3A and 3B, the fluorometer 102 includes first, second and third optical sources in the form of a first LED 106a, and second and third LEDs (not shown explicitly) for emitting first, second and third light respectively for illuminating the analyte 4, wherein the first light has a first spectral range, the second light has a second spectral range, and the third light has a third spectral range. The fluorometer 102 also includes a photodetector generally designated 110 including a silicon PIN photodiode 110a for detecting light output from the analyte 4 when the analyte 4 is illuminated by at least one of first, second and third light emitted by the first, second and third LEDs respectively and an amplifier 110b for amplifying an electrical signal generated by the silicon PIN photodiode 110a. The amplifier 110b is a multi-stage amplifier comprising a transimpedance stage and a gain stage.

[0391] 55789724-1FIG. 7C shows the first LED 106a and the PIN photodiode 110a secured relative to one another. The first LED 106a and the PIN photodiode 110a are arranged relative to one another such that the first LED 106a can emit first light along a first input direction and the PIN photodiode 110a can receive light along an output direction, wherein the output direction is generally orthogonal to the first input direction. The second and third LEDs are not shown in FIG. 7C in the interests of clarity, but it should be understood that the second and third LEDs are arranged to emit second and third light along second and third input directions respectively, wherein the output direction is also generally orthogonal to the second and third input directions. The fluorometer 102 further comprises one or more input optical components such as one or more input optical windows 144 through which light is coupled from the first LED 106a, and the second and third LEDs, to the analyte 4, and an output optical component such as an output optical window 148 through which light is coupled from the analyte 4 to the PIN photodiode 110a.

[0392] The fluorometer 102 further includes first, second and third LED drivers 108a, 108b, 108c for electrically driving the first, second and third LEDs respectively and a heatsink 170.

[0393] Like the fluorometer 2, the fluorometer 102 also includes a user interface 112, a memory (not shown), and a controller (not shown) in the form of a microcontroller. The memory stores calibration data.

[0394] As shown in FIGS. 7A and 7B, the fluorometer 2 further comprises a power supply in the form of a battery pack 122 and electronic circuitry for controlling the supply of power from the battery pack 122 to the first, second and third LED drivers, the photodetector 110, the user interface 112, the memory, and the controller. The user interface 112 comprises a display 112a, one or more indicator LEDs 112b, a user control in the form of a switch 112c for operating the fluorometer 102, and associated electronic circuitry.

[0395] The fluorometer 102 includes one or more circuit boards 140 such as one or more PCBs on which the first, second and third LEDs 106a, 106b and 106c, the first, second and third LED drivers 108a, 108b, 108c, the memory, the controller, the electronic circuitry associated with the power supply 122, the photodetector 110 including the silicon PIN photodiode 110a, the multi-stage amplifier 110b, and the electronic circuitry associated with the user interface 112 are mounted.

[0396] The fluorometer 102 further comprises a housing 126 which includes first, second, third and fourth parts 126a, 126b, 126c and 126d respectively, wherein the first

[0397] 55789724-1housing part 126a contains at least the first, second and third LEDs 106a, 106b and 106c and the silicon PIN photodiode 110a and is configured for dipping into the analyte 4 as shown in FIG. 7C. The first, second, third and fourth parts 126a, 126b, 126c and 126d are configured for connection to one another so as to enclose the first, second and third LEDs 106a, 106b and 106c, the first, second and third LED drivers 108a, 108b, 108c, the heatsink 170, the photodetector 110, the user interface 112, the memory, the controller, and the battery pack 122. The housing 126 is configured to act as a Faraday cage for at least partially suppressing any noise induced in the electrical signal which is generated by the photodetector 110 and transferred to the controller, for example due to any electromagnetic interference originating from an environment external to the second housing 126. Moreover, a hermetic seal is provided between the first part 126a of the housing 126 and the one or more input optical windows 144 and between the first part 126a of the housing 126 and the output optical window 148 to prevent ingress of the analyte 4 into the interior of the housing 126. From the foregoing description, one of skill in the art will understand that the first housing part 126a defines a pentahedral space or channel for the analyte 4 externally thereof with the input optical windows 144 and the output optical window 148 located on different sides of the pentahedral space or channel for the analyte 4such that the first LED 106a, the second and third LEDs (not shown in FIGS. 7A-7C), and the photodetector 110a are arranged at different sides of the pentahedral space or the channel for the analyte 4.

[0398] In use, the first housing part 126a of the fluorometer 102 is dipped or immersed into the analyte 4 as shown in FIG. 7C and the fluorometer 102 is operated via the switch 112c. In response to operation of the switch 112c, the controller of the fluorometer 102 controls the first, second and third LED drivers sequentially so that the first, second and third LEDs emit the first, second and third light sequentially, the controller causes the photodetector 110 to detect the resulting light output from the analyte 4, and the controller determines the concentrations of first, second and third constituents of the analyte 4 based on first, second and third electrical signals generated by the photodetector 110 and the calibration data stored in the memory in essentially the same way as described above in relation to the fluorometer 2. Moreover, it should be understood that the first LED 106a, the second and third LEDs (not shown in FIGS. 7A-7C), are only separated from the analyte 4 in the pentahedral space or channel by the thickness of the respective input optical windows 144. Similarly, the photodiode 110a is only separated from the analyte 4 in the pentahedral space or channel by the thickness of the output optical window 148. In effect, this arrangement allows the analyte 4 to act

[0399] 55789724-1as a heat sink for any heat generated by the first LED 106a, the second and third LEDs (not shown in FIGS. 7A-7C) thereby at least partially reducing the effects of heat on the spectra of the light output from the first LED 106a, the second and third LEDs (not shown in FIGS. 7A-7C) and / or at least partially reducing the effects of heat on the responsivity spectrum of the photodiode 110a. This may improve the stability and repeatability of the multispectral measurements performed using the fluorometer 102 from the time of the multispectral calibration procedure which is described with reference to FIGS. 4A to 5C and which is performed on a series of analytes of known composition to the time of the multispectral measurements performed on the analyte 4 of unknown composition. This may, in turn, improve the accuracy of the measurements of the concentrations of the first fluorescent constituent, the second fluorescent constituent, and the plurality of scattering particles of the analyte 4 of unknown composition using the multispectral measurement techniques described above.

[0400] One of ordinary skill in the art will also understand that various modifications are possible to the fluorometers 2, 102 and the associated methods for determining the concentrations of first, second and third constituents of an analyte described above. For example, although the methods described above comprise the measurement of the first, second and third electrical signals during sequential illumination of the analyte 4 with the first, second and third light 60a, 60b, 60c, the first, second and third light 60a, 60b, 60c may be modulated differently, and the first, second and third electrical signals may be measured during simultaneous illumination of the analyte 4 with the first, second and third light 60a, 60b, 60c if the photodetector 10 is configured to discriminate between the first, second and third electrical signals according to the different modulation of the first, second and third electrical signals resulting from use of the differently modulated the first, second and third light 60a, 60b, 60c. The first, second and third light 60a, 60b, 60c may, for example, be amplitude modulated at different frequencies.

[0401] Optionally, the first and second excitation spectral ranges do not overlap and the method comprises illuminating the analyte with first light having a first peak wavelength which is less than the first cut-off wavelength to cause the first fluorescent constituent to generate fluorescence in the analyte and resulting in scattering of the first light in the analyte, but without causing the second fluorescent constituent to generate fluorescence in the analyte. Put another way, the first spectral range may be selected so that the first electrical signal resulting from illumination of the analyte using the first light is dependent on the concentration of the first and third constituents but independent of, or substantially independent of, the concentration of the second constituent, the second spectral range

[0402] 55789724-1may be selected so that the second electrical signal resulting from illumination of the analyte using the second light is dependent on the concentration of the second and third constituents but independent of, or substantially independent of, the concentration of the first constituent, and the third spectral range may be selected so that the third electrical signal resulting from illumination of the analyte using the third light is dependent on the concentration of the third constituent but independent of, or substantially independent of, the concentrations of the first and second constituents. In these circumstances, the calibration procedure may be simplified so that the calibration data are derived from: measurements of the first electrical signal resulting from illumination using the first light of different known analytes having:

[0403] different known concentrations of the first constituent, and zero or insignificant concentrations of the second and third constituents;

[0404] different known concentrations of the third constituent, and zero or insignificant concentrations of the first and second constituents; measurements of the second electrical signal resulting from illumination using the second light of different known analytes having:

[0405] different known concentrations of the second constituent, and zero or insignificant concentrations of the first and third constituents; and

[0406] different known concentrations of the third constituent, and zero or insignificant concentrations of the first and second constituents; and measurements of the third electrical signal resulting from illumination using the third light of different known analytes having:

[0407] different known concentrations of the third constituent, and zero or insignificant concentrations of the first and second constituents.

[0408] Put another way, the first spectral range, the second spectral range, and the third spectral range may be selected relative to the first and second excitation spectral ranges of the first and second fluorescent constituents so that2i=3i= m32= mi2= 0 resulting in a simplified responsivity matrix M of the form:

[0409] mu 0 m13- M = 0 m22m23

[0410]

[0411] 0 m33.

[0412] In these circumstances, the calibration procedure may be simplified to determine only the non-zero responsivity coefficients of the simplified responsivity matrix M.

[0413] 55789724-1Although the PIN photodiodes 10a, 110a of the fluorometers 2, 102 are configured to receive light from the analyte 4 in an output direction which is generally orthogonal to the first, second and third input directions, the PIN photodiodes 10a, 110a may be configured to receive light from the analyte 4 in an output direction which is not orthogonal to the first, second and third input directions. For example, the PIN photodiodes 10a, 110a may be configured to receive light from the analyte 4 in an output direction arranged at an angle in the range of 85 to 95 degrees or in the range of 89 to 91 degrees, at an angle substantially equal to 90 degrees, or at an angle of 90 degrees relative to each of the first, second and third input directions.

[0414] In a variant of the optical assembly 30 of FIGS. 3A and 3B of the fluorometer 2, one or more of the primary input lenses 44, one or more of the secondary input lenses 46, and / or one or more of the output lenses 48 may be omitted. One of skill in the art will understand that one or more of the primary input lenses 44, one or more of the secondary input lenses 46, and / or one or more of the output lenses 48 may be omitted at least in part because the methods for determining the concentrations of first, second and third constituents of an analyte described above with reference to FIGS. 3A and 3B do not rely upon the use of any spectral filters between the primary input lenses 44, between the secondary input lenses 46, or between the output lenses 48. Moreover, omitting one or more of the primary input lenses 44 may allow the first and second LEDs 6a, 6b to be mounted relative to the container or cuvette 31 so that the first and second LEDs 6a, 6b are only separated from the analyte 4 by the thickness of the wall of the container or cuvette 31. Similarly, omitting one or more of the secondary input lenses 46 may allow the third LED 6c to be mounted relative to the container or cuvette 31 so that the third LED 6c is only separated from the analyte 4 by the thickness of the wall of the container or cuvette 31. In addition, omitting one or more of the output lenses 48 may allow the photodiode 10a to be mounted relative to the container or cuvette 31 so that the photodiode 10a is only separated from the analyte 4 by the thickness of the wall of the container or cuvette 31. In effect, this arrangement may allow the analyte 4 to act as a heat sink for any heat generated by the first, second and / or third LEDs 6a, 6b, 6c thereby at least partially reducing the effects of heat on the spectra of the light output from the LEDs 6a, 6b, 6c and / or at least partially reducing the effects of heat on the responsivity spectrum of the photodiode 10a. This may improve the stability and repeatability of the multispectral measurements performed using the fluorometer 2 from the time of the multispectral calibration procedure which is described with reference to FIGS. 4A to 5C and which is performed on a series of analytes of known composition to

[0415] 55789724-1the time of the multispectral measurements performed on the analyte 4 of unknown composition. This may, in turn, improve the accuracy of the measurements of the concentrations of the first fluorescent constituent, the second fluorescent constituent, and the plurality of scattering particles of the analyte 4 of unknown composition using the multispectral measurement techniques described above. In the fluorometer 2 described with reference to FIGS. 2A-3B, the container or cuvette 31 has four sides when viewed in plan, the first and second LEDs 6a, 6b illuminate the analyte 4 in the container or cuvette 31 through a first side of the container or cuvette 31, the third LED 6c illuminates the analyte 4 in the container or cuvette 31 through a second side of the container or cuvette 31 opposite the first side of the container or cuvette 31, and the photodetector 10a detects light such as fluorescence or scattered light emanating through a third side of the container or cuvette 31. In other fluorometer embodiments, the container or cuvette 31 may have three sides when viewed in plan, the first and second LEDs 6a, 6b may illuminate the analyte 4 in the container or cuvette 31 through a first side of the container or cuvette 31, the third LED 6c may illuminate the analyte 4 in the container or cuvette 31 through a second side of the container or cuvette 31, and the photodetector 10a may detect light such as fluorescence or scattered light emanating through a third side of the container or cuvette 31. In yet further fluorometer embodiments, the container or cuvette 31 may have five or more sides when viewed in plan, each of the first, second, and third LEDs 6a, 6b, 6c may illuminate the analyte 4 in the container or cuvette 31 through a different input sides of the container or cuvette 31, and the photodetector 10a may detect light such as fluorescence or scattered light emanating through an output side of the container or cuvette 31 which is not opposite any of the input sides of the container or cuvette 31.

[0416] In the fluorometer 102 described with reference to FIGS. 7A-7C, the first housing part 126a defines a pentahedral space or channel for the analyte 4 externally thereof with the input optical windows 144 and the output optical window 148 located on different sides of the pentahedral space or channel for the analyte 4 and with the first LED 106a, the second and third LEDs (not shown in FIGS. 7A-7C), and the photodetector 110a arranged at different sides of the pentahedral space or the channel for the analyte 4. In other fluorometer embodiments, the fluorometer housing may define a space or channel for the analyte 4 which may have a shape other than a pentahedral shape with the first, second and third LEDs and the photodetector arranged at the sides of the space or the channel for the analyte 4. For example, the fluorometer housing may define a space or a channel for the analyte which has a V-shape or a tetrahedral shape.

[0417] 55789724-1Although a fluorometer and associated methods for determining the composition of an analyte comprising first, second and third constituents are described in terms of specific embodiments as set forth above, it should be understood that these embodiments are illustrative only and that the invention is not limited to those embodiments. Those skilled in the art will be able to make modifications to the described embodiments and / or to identify alternatives to the described embodiments which fall within the scope of the appended claims in view of the disclosure. Each feature disclosed or illustrated herein may be incorporated in any embodiment, whether alone or in any appropriate combination with any other feature disclosed or illustrated herein. In particular, one of ordinary skill in the art will understand that one or more of the features of the embodiments of the present disclosure described above with reference to the drawings may produce effects or provide advantages when used in isolation from one or more of the other features of the embodiments of the present disclosure and that different combinations of the features are possible other than the specific combinations of features of the embodiments of the present disclosure described above.

[0418] The skilled person will understand that in the preceding description and appended claims, positional terms such as ‘above’, ‘along’, ‘side’, etc. are made with reference to the accompanying drawings. These terms are used for ease of reference but are not intended to be of limiting nature. These terms are therefore to be understood as referring to an object when in an orientation as shown in the accompanying drawings.

[0419] Use of the term "comprising" when used in relation to a feature of an embodiment of the present disclosure does not exclude other features or steps. Use of the term "a" or "an" when used in relation to a feature of an embodiment of the present disclosure does not exclude the possibility that the embodiment may include a plurality of such features.

[0420] The use of reference signs in the claims should not be construed as limiting the scope of the claims.

[0421] 55789724-1

Claims

CLAIMS1. A fluorometer for use in determining the concentrations of first, second and third constituents of an analyte, wherein the first constituent comprises a first fluorescent constituent, the second constituent comprises a second fluorescent constituent, and the third constituent comprises a plurality of scattering particles, and wherein the fluorometer comprises:first, second and third optical sources for emitting first, second and third light with first, second and third spectral ranges respectively;a photodetector for detecting light output from the analyte when the analyte is illuminated by at least one of the first, second and third light; anda controller,wherein the controller is configured to:cause the first optical source to illuminate the analyte using the first light and cause the photodetector to detect light output from the analyte so as to generate a first electrical signal resulting from illumination of the analyte using the first light;cause the second optical source to illuminate the analyte using the second light and cause the photodetector to detect light output from the analyte so as to generate a second electrical signal resulting from illumination of the analyte using the second light;cause the third optical source to illuminate the analyte using the third light and cause the photodetector to detect light output from the analyte so as to generate a third electrical signal resulting from illumination of the analyte using the third light; anddetermine a concentration of each of the first, second and third constituents based at least in part on the first, second and third electrical signals.

2. The fluorometer as claimed in claim 1, comprising a memory storing calibration data, wherein the controller is configured to determine the concentration of each of the first, second and third constituents based on the first, second and third electrical signals in combination with the stored calibration data, and optionallywherein the calibration data comprises responsivity coefficientsand offset coefficients b^, wherein m7and bijare obtained by measuring the electrical signals s7= rriij Cj + bij for known concentrations cjof each constituent j excited by light i during a55789724-1calibration procedure and using a regression analysis technique such as a linear regression analysis technique, a Multivariate Linear Regression analysis technique, ora Bayesian regression analysis technique, to fit the measured electrical signals stj as a function of the known concentrations cjof each constituent j, wherein i = 1,2,3 and j = 1,2,3.

3. The fluorometer as claimed in claim 1 or 2, wherein at least one of:there are no spectral filters between the first optical source and the analyte; there are no spectral filters between the second optical source and the analyte; there are no spectral filters between the third optical source and the analyte; or there are no spectral filters between the analyte and the photodetector.

4. The fluorometer as claimed in any preceding claim, comprising a housing, wherein the housing defines an interior space internally thereof for housing the first, second and third optical sources, the photodetector, the memory, and the controller, and wherein at least one of:the housing defines an opening or an aperture for inserting a container for containing the analyte into the interior space and / or for inserting a flow cell or fluid conduit for conducting a flow of the analyte into the interior space, or the housing is configured so that the interior space is sealed from an environment exterior to the housing, and optionally wherein, the housing comprises a dipping section for dipping into the analyte, wherein the dipping section is configured to separate the first, second and third optical sources and the photodetector from the analyte when the dipping section is dipped into the analyte;the housing defines a space or a channel for the analyte externally thereof; the first, second and third optical sources and the photodetector are arranged around the space or the channel for the analyte;the first, second and third optical sources and the photodetector are arranged at one or more sides of the space or the channel for the analyte;the first, second and third optical sources and the photodetector are arranged at different sides of the space or the channel for the analyte;the space or the channel has a V-shape, a tetrahedral shape, or a pentahedral shape; orthe housing comprises a handle portion or is configured to be handheld.55789724-15. The fluorometer as claimed in any preceding claim, wherein at least one of: at least two of the first, second and third optical sources are arranged to enable said at least two of the first, second and third optical sources to illuminate the analyte along the same input optical axis;at least two of the first, second and third optical sources are arranged side-by-side to enable said at least two of the first, second and third optical sources to illuminate the analyte along respective parallel input optical axes;at least two of the first, second and third optical sources are located at different positions to enable said at least two of the first, second and third optical sources to illuminate the analyte along respective non-parallel input optical axes;at least two of the first, second and third optical sources are arranged on opposite sides of the analyte;at least two of the first, second and third optical sources are arranged side-by-side on the same side of the analyte;the first optical source is arranged to illuminate the analyte along a first input direction, the second optical source is arranged to illuminate the analyte along a second input direction, the third optical source is arranged to illuminate the analyte along a third input direction, and the photodetector is arranged to receive light output from the analyte along an output direction, wherein the output direction is arranged at a non-zero angle relative to each of the first, second and third input directions;the output direction is arranged at an angle in the range of 85 to 95 degrees or in the range of 89 to 91 degrees, at an angle substantially equal to 90 degrees, or at an angle of 90 degrees relative to each of the first, second and third input directions; or at least two of the first, second and third input directions are the same.

6. The fluorometer as claimed in any preceding claim, wherein one or more of the first, second and third optical sources comprises an LED or a laser diode.

7. The fluorometer as claimed in any preceding claim, wherein at least one of: the photodetector comprises or is a photodiode;the photodetector comprises or is a single photodiode;the photodetector comprises or is a silicon photodiode;the photodetector comprises or is a PIN photodiode; orthe photodetector comprises or is a single pixel photodiode.55789724-18. The fluorometer as claimed in claim 7, wherein the photodetector comprises an amplifier for amplifying an electrical signal generated by the photodiode, and optionally wherein at least one of:the amplifier comprises a multi-stage amplifier;the amplifier comprises a transimpedance stage and a gain stage; the amplifier comprises a lock-in amplifier;the amplifier is configured to amplify electrical signals in a frequency range which includes the modulation frequency or frequencies of the first, second and third light;the amplifier is configured to only amplify electrical signals in a frequency range which includes the modulation frequency or frequencies of the first, second and third light;the amplifier is configured to amplify electrical signals in a frequency range of 250 Hz to 5 KHz; orthe amplifier is configured to only amplify electrical signals in a frequency range of 250 Hz to 5 KHz.

9. The fluorometer as claimed in claim 8, wherein at least one of:the controller is configured to modulate the first light emitted by the first optical source and to cause the amplifier to use lock-in detection to generate the first electrical signal resulting from illumination of the analyte using the first light;the controller is configured to modulate the second light emitted by the second optical source and to cause the amplifier to use lock-in detection to generate the second electrical signal resulting from illumination of the analyte using the second light; or the controller is configured to modulate the third light emitted by the third optical source and to cause the amplifier to use lock-in detection to generate the third electrical signal resulting from illumination of the analyte using the third light.

10. The fluorometer as claimed in any preceding claim, comprising a first Faraday cage, wherein the first Faraday cage encloses the photodetector, and optionally wherein the fluorometer comprises a second Faraday cage, wherein the second Faraday cage encloses at least the first Faraday cage, the first, second and third optical sources, the controller, and the memory.

11. The fluorometer as claimed in any preceding claim, comprising at least one of:55789724-1one or more primary input optical elements between the first optical source and the analyte, wherein the one or more primary input optical elements are configured to couple the first light from the first optical source to the analyte;one or more secondary input optical elements between the second optical source and the analyte, wherein the one or more secondary input optical elements are configured to couple the second light from the second optical source to the analyte; or one or more tertiary input optical elements between the third optical source and the analyte, wherein the one or more tertiary input optical elements are configured to couple the third light from the third optical source to the analyte, and optionally wherein at least one of:the one or more primary input optical elements comprise one or more primary lenses;the one or more secondary input optical elements comprise one or more secondary lenses;the one or more tertiary input optical elements comprise one or more tertiary lenses;the one or more primary input optical elements comprise one or more primary windows;the one or more secondary input optical elements comprise one or more secondary windows;the one or more tertiary input optical elements comprise one or more tertiary windows;the one or more primary windows are the only input optical elements between the first optical source and the analyte;the one or more secondary windows are the only input optical elements between the second optical source and the analyte; orthe one or more tertiary windows are the only input optical elements between the third optical source and the analyte.

12. The fluorometer as claimed in any preceding claim, comprising one or more output optical elements between the analyte and the photodetector, wherein the one or more output optical elements are configured to couple light output from the analyte to the photodetector, and optionally wherein at least one of:the one or more output optical elements comprise one or more output lenses;55789724-1the one or more output optical elements comprise one or more output windows; orthe one or more output windows are the only output optical elements between the analyte and the photodetector.

13. The fluorometer as claimed in any preceding claim, comprising a battery for supplying electrical power to at least one of the first, second and third optical sources, the photodetector, the memory, and the controller, and optionally, wherein the battery is rechargeable, and optionally, wherein the fluorometer comprises a generator arrangement for recharging the battery, and optionally, wherein the generator arrangement comprises a solar cell or a solar panel.

14. A method for use in determining the concentrations of first, second and third constituents of an analyte, wherein the first constituent comprises a first fluorescent constituent, the second constituent comprises a second fluorescent constituent, and the third constituent comprises a plurality of scattering particles, and wherein the method comprises:illuminating the analyte using first light having a first spectral range and using a photodetector to detect light output from the analyte so as to generate a first electrical signal resulting from illumination of the analyte using the first light;illuminating the analyte using second light having a second spectral range and using the photodetector to detect light output from the analyte so as to generate a second electrical signal resulting from illumination of the analyte using the second light;illuminating the analyte using third light having a third spectral range and using the photodetector to detect light output from the analyte so as to generate a third electrical signal resulting from illumination of the analyte using the third light; and determining a concentration of each of the first, second and third constituents based at least in part on the first, second and third electrical signals.

15. The method as claimed in claim 14, wherein determining the concentration of each of the first, second and third constituents based at least in part on the first, second and third electrical signals comprises determining the concentration of each of the first, second and third constituents based on the first, second and third electrical signals in combination with calibration data.55789724-116. The method as claimed in claim 15, wherein the calibration data comprises responsivity coefficients m7and offset coefficients b^, wherein m7and bijare obtained by measuring the electrical signals s7= m7cj+ bijfor known concentrations cjof each constituent j excited by light i during a calibration procedure and using a regression analysis technique such as a linear regression analysis technique, a Multivariate Linear Regression analysis technique, or a Bayesian regression analysis technique, to fit the measured electrical signals s7as a function of the known concentrations cjof each constituent j, wherein i = 1,2,3 and j = 1,2,3.

17. The method as claimed in claim 16, wherein determining the concentration of each of the first, second and third constituents based on the first, second and third electrical signals and the calibration data comprises determining the concentration of each of the first, second and third constituents cjaccording to:c = M-1(s — b)wherein c is a concentration vector given by:CTC2,c3.s is an electrical signal vector given by:-si-S2,s3.siis the measured ithelectrical signal generated by the photodetector resulting from illumination of the analyte using light i, M-1is the inverse of a responsivity matrix M given by:m11m12m13m21m22m23m31m32m33b is the offset vector given by:55789724-1Vb = b̄₂b̄₃and b̄i=bij / 3 is the mean of the offset coefficientsfor the first, second and third constituents.

18. The method as claimed in claim 16 or 17, wherein the calibration data are derived from:measurements of the first electrical signal resulting from illumination using the first light of different known analytes having:different known concentrations of the first constituent, and zero or insignificant concentrations of the second and third constituents;different known concentrations of the second constituent, and zero or insignificant concentrations of the first and third constituents; anddifferent known concentrations of the third constituent, and zero or insignificant concentrations of the first and second constituents; measurements of the second electrical signal resulting from illumination using the second light of different known analytes having:different known concentrations of the first constituent, and zero or insignificant concentrations of the second and third constituents;different known concentrations of the second constituent, and zero or insignificant concentrations of the first and third constituents; anddifferent known concentrations of the third constituent, and zero or insignificant concentrations of the first and second constituents; and measurements of the third electrical signal resulting from illumination using the third light of different known analytes having:different known concentrations of the first constituent, and zero or insignificant concentrations of the second and third constituents;different known concentrations of the second constituent, and zero or insignificant concentrations of the first and third constituents; anddifferent known concentrations of the third constituent, and zero or insignificant concentrations of the first and second constituents.

19. The method as claimed in claim 16 or 17, wherein the first spectral range, the second spectral range, and the third spectral range are selected relative to the first and55789724-1second excitation spectral ranges of the first and second fluorescent constituents so that m21= m31= m32= 0 i.e. so that:m11m12m13M = 0 m22m230 m33and / orwherein the first spectral range is selected so that the first electrical signal resulting from illumination of the analyte using the first light is dependent on the concentration of the first, second and third constituents, the second spectral range is selected so that the second electrical signal resulting from illumination of the analyte using the second light is dependent on the concentration of the second and third constituents but is independent of, or substantially independent of, the concentration of the first constituent, and the third spectral range is selected so that the third electrical signal resulting from illumination of the analyte using the third light is dependent on the concentration of the third constituent but independent of, or substantially independent of, the concentrations of the first and second constituents, andwherein the calibration data are derived from:measurements of the first electrical signal resulting from illumination using the first light of different known analytes having:different known concentrations of the first constituent, and zero or insignificant concentrations of the second and third constituents;different known concentrations of the second constituent, and zero or insignificant concentrations of the first and third constituents; anddifferent known concentrations of the third constituent, and zero or insignificant concentrations of the first and second constituents; measurements of the second electrical signal resulting from illumination using the second light of different known analytes having:different known concentrations of the second constituent, and zero or insignificant concentrations of the first and third constituents; anddifferent known concentrations of the third constituent, and zero or insignificant concentrations of the first and second constituents; and measurements of the third electrical signal resulting from illumination using the third light of different known analytes having:55789724-1different known concentrations of the third constituent, and zero or insignificant concentrations of the first and second constituents.

20. The method as claimed in claim 16 or 17, wherein the first spectral range, the second spectral range, and the third spectral range are selected relative to the first and second excitation spectral ranges of the first and second fluorescent constituents so that m21= m31= m32= m12= 0 i.e. so that:m110 m130 m22m230 0 m33.and / orwherein the first spectral range is selected so that the first electrical signal resulting from illumination of the analyte using the first light is dependent on the concentration of the first and third constituents but independent of, or substantially independent of, the concentration of the second constituent, the second spectral range is selected so that the second electrical signal resulting from illumination of the analyte using the second light is dependent on the concentration of the second and third constituents but independent of, or substantially independent of, the concentration of the first constituent, and the third spectral range is selected so that the third electrical signal resulting from illumination of the analyte using the third light is dependent on the concentration of the third constituent but independent of, or substantially independent of, the concentrations of the first and second constituents, andwherein the calibration data are derived from:measurements of the first electrical signal resulting from illumination using the first light of different known analytes having:different known concentrations of the first constituent, and zero or insignificant concentrations of the second and third constituents;different known concentrations of the third constituent, and zero or insignificant concentrations of the first and second constituents; measurements of the second electrical signal resulting from illumination using the second light of different known analytes having:different known concentrations of the second constituent, and zero or insignificant concentrations of the first and third constituents; and55789724-1different known concentrations of the third constituent, and zero or insignificant concentrations of the first and second constituents; and measurements of the third electrical signal resulting from illumination using the third light of different known analytes having:different known concentrations of the third constituent, and zero or insignificant concentrations of the first and second constituents.

21. The method as claimed in any one of claims 14 to 20, wherein the steps of: illuminating the analyte using first light and using the photodetector to detect light output from the analyte so as to generate a first electrical signal resulting from illumination of the analyte using the first light;illuminating the analyte using second light and using the photodetector to detect light output from the analyte so as to generate a second electrical signal resulting from illumination of the analyte using the second light; andilluminating the analyte using third light and using the photodetector to detect light output from the analyte so as to generate a third electrical signal resulting from illumination of the analyte using the third light,are performed sequentially.

22. The method as claimed in any one of claims 14 to 21, wherein at least one of:the first light is modulated and lock-in detection is used to generate the first electrical signal resulting from illumination of the analyte using the first light;the second light is modulated and lock-in detection is used to generate the second electrical signal resulting from illumination of the analyte using the second light; orthe third light is modulated and lock-in detection is used to generate the third electrical signal resulting from illumination of the analyte using the third light.

23. The fluorometer or the method as claimed in any preceding claim, wherein the analyte is, or comprises, a liquid, and / or wherein the analyte comprises a base constituent such as water, and the first, second and third constituents comprise first, second and third contaminants of the base constituent respectively.

24. The fluorometer or the method as claimed in any preceding claim, wherein at least one of:55789724-1the light output from the analyte when the analyte is illuminated using the first light comprises fluorescence generated by the first fluorescent constituent and scattered first light, and optionally also fluorescence generated by the second fluorescent constituent;the light output from the analyte when the analyte is illuminated using the second light comprises fluorescence generated by the second fluorescent constituent and scattered second light, and optionally also fluorescence generated by the first fluorescent constituent;the light output from the analyte when the analyte is illuminated using the third light comprises scattered third light, and optionally also fluorescence generated by the first and / or second fluorescent constituents;the first spectral range overlaps or falls within a first excitation spectral range of the first fluorescent constituent;the second spectral range overlaps or falls within a second excitation spectral range of the second fluorescent constituent;the third spectral range falls outside the first and second excitation spectral ranges; orthe second spectral range partially overlaps or falls outside the first excitation spectral range of the first fluorescent constituent.

25. The fluorometer or the method as claimed in any preceding claim, wherein the first spectral range includes a first peak wavelength, the second spectral range includes a second peak wavelength, and the third spectral range includes a third peak wavelength and optionally wherein at least one of:the first peak wavelength falls within the first excitation spectral range, the second peak wavelength falls within the second excitation spectral range, and the third peak wavelength falls outside the first and second excitation spectral ranges;the second peak wavelength falls inside or outside the first excitation spectral range of the first fluorescent constituent;the first peak wavelength is shorter than the second peak wavelength, and the second peak wavelength is shorter than the third peak wavelength;the first excitation spectral range of the first fluorescent constituent has a first cutoff wavelength such that when the first fluorescent constituent is excited using light at wavelengths which are longer than the first cut-off wavelength, the first fluorescent constituent generates little or no fluorescence;55789724-1the second excitation spectral range of the second fluorescent constituent has a second cut-off wavelength such that when the second fluorescent constituent is excited using light at wavelengths which are longer than the second cut-off wavelength, the second fluorescent constituent generates little or no fluorescence;the first and second excitation spectral ranges overlap in an overlap excitation spectral range at wavelengths less than the first cut-off wavelength and the first light has a first peak wavelength which is in the overlap excitation spectral range to cause both the first and second fluorescent constituents to generate fluorescence in the analyte and resulting in scattering of the first light in the analyte, or the first and second excitation spectral ranges do not overlap and the first light has a first peak wavelength which is less than the first cut-off wavelength to cause the first fluorescent constituent to generate fluorescence in the analyte and resulting in scattering of the first light in the analyte, but without causing the second fluorescent constituent to generate fluorescence in the analyte;the second light has a second peak wavelength which is between the first and second cut-off wavelengths; orthe third light has a third peak wavelength which is greater than the second cutoff wavelength.

26. The fluorometer or the method as claimed in any preceding claim, wherein at least one of:the first fluorescent constituent comprises a Tryptophan-like fluorescence (TLF)-emitting constituent;the first spectral range is selected to excite TLF in the first fluorescent constituent; the first spectral range includes a peak TLF excitation wavelength;the second fluorescent constituent comprises a humic-like fluorescence (HLF)-emitting constituent;the second spectral range is selected to excite HLF in the second fluorescent constituent;the second spectral range includes a peak HLF excitation wavelength;the first spectral range is selected to excite TLF in the first fluorescent constituent and HLF in the second fluorescent constituent;the second spectral range is selected to excite HLF in the second fluorescent constituent but not to excite TLF in the first fluorescent constituent; or55789724-1the third spectral range is selected to fall outside an excitation spectral range of TLF and outside an excitation spectral range of HLF.

27. The fluorometer or the method as claimed in any preceding claim, wherein at least one of:the first peak wavelength comprises a wavelength in the range of 265 to 295 nm, in the range of 275 to 285 nm, a wavelength of 280 nm or substantially equal to 280 nm, or a wavelength of 275 nm or substantially equal to 275 nm;the second peak wavelength comprises a wavelength in the range of 345 to 385 nm, in the range of 355 to 375 nm, or a wavelength of 365 nm or substantially equal to 365 nm;the third peak wavelength comprises a wavelength in the range of 500 to 1,100 nm, in the range of 900 to 1,000 nm, or a wavelength of 940 nm or substantially equal to 940 nm; orthe third peak wavelength comprises a wavelength in the range of 750 to 1,100 nm, in the range defined by the ISO:7027 specification, and / or in the range of 830 to 890 nm.55789724-1

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